Hi my name's David Julius, I'm in the Physiology Department
at UC San Francisco. And I'd like to take a few minutes to tell you
about how we have used spices to understand mechanisms
for temperature and pain sensation. And I thought I'd begin
just by telling you a little bit about things that have interested me
and what sort of inspired me to work in this area.
So for many years, I've had an interest in natural products
and how these can be used to explore physiology and neuropharmacology.
And to wonder how people have discovered the use of plants
to folk medicine and other ritualistic behaviors. How the
active ingredients were identified and how they've been used
in biology to explore signaling pathways. And I just put
this slide together to show you some of my heroes in this area.
And how they've used natural products from things from opium poppies,
psilocybin, or marijuana to understand key signaling pathways
in the brain, like opiate receptors, serotonin receptors,
and cannabinoid receptors. And to illustrate the poewr of natural products
for identifying endongenous signaling components in a system.
The other thing that I've been interested in for many years
like many people in science, is to understand sensory systems.
And I think for many of us, including myself, this fascination
really comes from the idea that sensory systems really determine
how we perceive our world. The kinds of frequencies of light we can see,
the sounds -- the frequencies of sound we can hear, the
things that we can taste, are really determined entirely
by the sorts of receptors that we have in our sensory system and how
that information is integrated in our neural circuits. And
what's also interesting is that different animals see the world
in different ways, because they have different receptors.
A rattlesnake, for example, can see a rodent on a moonless night.
Because it can detect its body heat, and so I think this raises
fascinating questions about evolution and about how biophysics has
tuned sensory systems to enable us to live in the environment
that we need to live in. Some years ago, we decided to merge
these interests and to study an aspect of sensory systems that
at the time was less well understand, than say chemosensor
systems like olfaction, taste, and certainly vision.
And that is to understand somatosensation, or sensations of touch,
in particular, to understand this in relationship to how we experience
pain. And the aspect of pain sensation that we focused on
is nociception, which is the initial act of detecting noxious stimulants. So
nociception is to pain, for example, what phototransduction is to vision.
And we've been interested in this subclass of sensory nerve
fibers called C and A-delta fibers, the so-called nociceptors
that are the subset of somatosensory neurons that carry
out the bulk of detection of noxious stimuli that contribute to pain
sensation. And our goal was really to identify the molecules and
mechanisms that enable these nerve fibers to detect
things like environmental stimuli, changes in temperature
or pressure, or noxious chemicals, or even endogenous
things that are produced during inflammation. Endogenous
chemicals like prostaglandins and other things that generate
or contribute to pain sensation. And the place where this all
comes together for me is really that natural products have played such a
key role in trying to understand how these nerve fibers work.
And probably the best example is given by pungent agents from
capsicum peppers, or hot chili peppers. And back in the 70s, Jansco and
colleagues in Hungary showed that the pungent ingredient in hot chili
peppers, mainly capsaicin, illicits a sensation of burning pain.
By serving as a specific chemical activator of these nerve fibers that are
involved in pain reception, mainly the nociceptors. And some years
later, Bevan, Rang, and others in London carried out some biophysical studies
to suggest that what capsaicin does when it interacts with nerve fibers
in your mouth or anywhere else in your somatotopic surface,
is to depolarize the nerve fiber and to allow sodium, calcium ions
to flow in. And the main questions that we're still unresolved
by this was, does capsaicin interact with a specific site, a so-called
vanilloid site? Because capsaicin is a vanilloid compound,
like other pungent agents in peppers. Is there a specific receptor
for capsaicin? If so, what does it look like? And what might its normal
physiologic function be in the body? What role does it serve
in our endogenous ability to sense pain? And we, like many other
people in the field, realized identifying or addressing these questions
could lead to some key insights into pain sensation, because
sensitivity to capsaicin was a major functional hallmark
or characteristic of these nerve fibers that are involved in
making this sensation. In many ways, this was to the nociceptor
what the T-cell receptor was to lymphocytes. And so we set out
to do this, but the problem was that nobody really knew what
this receptor might look like, even if it existed. And so we decided
to take sort of a structure-blind identity-blind approach to doing
this. By just exploiting some functional attributes. And the idea
was that, if the capsaicin receptor does exist, you could make a cDNA library
from sensory nerve ganglia, and introduce those clones into
a non-neural cell that's normally capsaicin-insensitive,
such as a fibroblast. And then if you expose these to capsaicin
and that allowed calcium to flow into the cell or somehow be
released from extracellular stores, you could then detect this
using calcium-sensitive dyes, such as those developed by Roger Tsien and
all those like Indo or Fura. This has inherent risks, the advantage
is that you make no predispositions about what this receptor,
mythical receptor, might look like. But the risk is that if the
receptor is encoded by multiple gene products, the ability
to transfer a single gene to a fibroblast and recapitulate this activity
would be very difficult, if not impossible. But you know, you can't do
science without luck and some risk, and that's kind of what we're all
in this business for. In some ways, to do something new and exciting.
And so we tried this and low and behold, we ended up by this method
of identifying a single cDNA that encoded for a capsaicin receptor,
which we called the vanilloid receptor. And this is work done by
a wonderful postdoc in my lab at the time, Mike Caterina,
who now has his own group at Johns Hopkins University.
And we could show that this cloned gene encoded a functional
receptor, because if you introduce it into these cells, and use
patch clamp electrophysiology to look at what's happening,
we could show that when you put on capsaicin into these
cells, in fact you induce inward current flow of sodium and calcium
ions into the cell. And so this told us that the TRPV1 that
we had identified, this gene, is in fact a vanilloid activated channel.
But Makoto Tominaga and Mike Caterina and others in my lab then
addressed really the most interesting question, which is what
else does this receptor respond to and what might its normal
physiological role be in our ability to sense noxious stimuli?
And we tried to activate this receptor with all kinds of things
that we knew generated pain psychophysically or activated
sensory neurons in culture or nerve fibers. And we went through a whole
gamut of things, neuropeptides, other neurotransmitters.
And it was only after some time that we realized, gee these
nerve fibers detect changes in our environment, as well as
changes internally that relate to pain sensation. And we should try
some of these things. And the one that we tried that worked was
heat. And in retrospect, this seems obvious because you describe
a chili pepper often as a hot chili pepper. And this is a very nice
parsimonious explanation of that. But this wasn't the first thing that
we thought about, but you know, shortly after failing to look at it
-- to find results in the other stimuli, it occurred to us that this might
be the case. And it's really a beautiful result, in that you see that
the channel, when expressed in this case, in a frog oocyte, is silent
until you heat the solution, the external solution up, and then the channel
opens, and you see this large ionic current go through the cell
with a threshold that's very consistent with the sensitivity
of heat-sensitive nociceptors to heat. And in fact, is very close
to the threshold that most of us would describe as the demarcation
point between determining if something is innocuously warm
and noxiously hot. So this has led us to propose that TRPV1
is a little molecular thermometer that sits at the membrane, and
at least contributes, maybe doesn't underlie all, but contributes
to our ability to detect ambient temperature in the warm and the hot
range. Now we all know that we can sense temperatures over a
wide range, including cold, not just hot. And how might this
work? We thought that if we could figure this out, we could maybe
get some larger view of how temperature sensation works.
And ask if our ability to sense temperatures of different degrees
is due to a sort of similar, unifying molecular concept or
represents different molecular mechanisms. And again, we wanted
to take an approach that was not biased. We didn't want
to look for channels and genes that were similar to TRPV1,
but instead, step back and ask how is cold sensed
from just a functional point of view. And again, the great way
here, we turned to natural products. Because we all know
that cooling agents that are derived from mint leaves or eucalyptol,
produces a sensation of cooling. And Hensel and Zotterman
in the '50s in Sweden showed that this was the case in neurophysiological
recordings of sensory nerve fibers. So we played the same game
that we played with capsaicin to ask if we could identify
a menthol receptor. And the question was, is there a menthol
receptor? If so, does it respond to cold? And is it related to TRPV1?
Or is this a totally independent molecular mechanism?
And so we did, meaning David McKemy, a postdoc in my lab
at the time, and Werner Neuhausser, ended up identifying
a clone and a molecule called TRPM8, that when expressed
in fibroblasts, renders them sensitive to menthol and
other cooling agents. And as you can tell from the name, TRPM8,
it belongs to the same family of so-called TRP ion channels,
as does TRPV1, it's a molecular cousin. And the question is,
does it respond to cold? And in fact, again, when you express this
in a frog oocyte or some other cells, you can see that the channel is
quiet at warm temperatures, but then opens when you cool the
cell below about 26 degrees. And so this led to this idea that
there is some unifying concept of how we sense changes in
ambient temperature, and TRP channels serve as, or at least contribute
to our ability to detect changes in ambient temperature over a pretty
wide range. And they act as little molecular thermometers in
membrane that initiate the polarization of the neural fiber
in response to changes in temperature. And the best way really to test this,
of course, is in a living animal with genetics. Using genetics.
And I think the clearest demonstration of this really comes
from knock outs of TRPM8, which show a very beautiful phenotype
in terms of deficits and thermosensitivities. So we made a mouse
that lacks this channel, this is work done by these wonderful
people in my lab, Jans Siemens, Diana Bautista, and Sven Jordt, who now
all have their own labs in Heidelberg, and UC Berkeley, and
Duke University, respectively. And they set up a very nice
simple paradigm to look and see if deletion of this gene has a
phenotype associated with it, with temperature sensitivity. So
here you do this very simple test, where you take in this case, a wild type
mouse. And you ask if it can discriminate between a warm
platform and one that's somewhat cooler. Namely 20 and 30 degrees.
And you can see without any training, this animal avoids the cool
side and likes to stay on the nice, warm, cozy side. As is true for most
of us. And he'll stay on this side all day long. Now
if instead, you play this game with a mouse that, in which the gene
for the menthol receptor is knocked out, TRPM8 deficient mouse,
you'll see that the animal cannot discriminate between these two
platforms. And will wander, will spend equal time on each
of them. In fact, it won't be able to discriminate between platforms
even when the cool side goes down to very low temperatures,
to about 10 degrees. Below that, the animal can detect changes,
can detect the preference for the warm side, which may be due
to other processes that contribute to cold sensation. But in any case,
I think this validates the idea that TRP channels, and in particular,
TRPM8, play a really important role as a molecular detector
of ambient temperature. So through these studies, we've
been able to identify key elements that enable cells, that
really enable cells to specify their sensitivity to environmental stimuli.
And as in other sensory systems, the great things about doing this,
identifying genes and molecules that specify the sensitivity
of cells and nerve fibers to stimuli, is that you can use them as
molecular probes to try and understand the cellular and circuit
logic, as to how this information is conveyed to the nervous system
and ultimately, to your brain. So that you can discriminate different
percepts, in this case, heat from cold. And what these markers,
antibodies to these channels now tell us, is that the cold and
hot receptors are really segregated. They're expressed by
distinct subpopulations of sensory nerve fibers. Which tells us
at least, at the offset that the information as it comes into the spinal
chord, at least at the beginning, is segregated. And that information about cold
and information about heat, is conveyed by two so-called labeled lines.
Separate lines of sensory nerve fibers. And this at least begins to give
us some idea as to how behavioral specificity is dictated
by the circuitry in at least a peripheral aspect of the system.
So, we've used natural products to identify some important
players in pain sensation, some of which I haven't had time to
tell you about today. And all of this work has really come
about through just sort of curiosity driven research, which by the way
was funded by you, the taxpayers, via the NIH. And we've just
been curious about how the system works and allows us to
appreciate the world around us. But as so often the case, curiosity
driven research can have very practical and important
therapeutics aspects. And all these channels are now novel
targets through the development, hopefully, of new types of
analgesic drugs that we can use to treat a variety of peripheral pain
syndromes. And that's work that's in progress by a number of
pharmaceutical and academic labs. But hopefully, our goal
to understand how this system works, and the curiosity-based
level, will also have some practical outcome that will benefit
the patients who suffer from common pain syndromes.
And finally, I'd like to say that, my lab is small. There are maybe
about -- or relatively small, anywhere between 7 and 9 people
at any given time. And so the way that we cover the waterfront,
I'm a big believer in trying to hit a problem from multiple
different angles, and in my lab, we try to do this starting from
molecular biology through biophysics, through animal behavior
and anatomy, and physiology, and ultimately structural
biology. And the way that we do this is to work with other fantastic
scientists, through close collaborations, some at my own institutions,
some other places in the world. And this has really allowed us to
expand our scientific horizons, which is something I think every
scientist is trying to do. And to work outside the comfort
zone, and to stretch yourself. And of course, all these people
are now great friends. And this is a wonderful way to meet people
and have life-long friends. And also enlarge the sphere of people
who focus on what your lab can interact with. And so, I'm a big believer
in collaborative science. And we've had a fantastic time working
on these projects within my lab and with friends around the world.
Thank you very much.