Hi. My name
is Adam Cohen. I am a professor
in the Departments of Chemistry
and Physics at Harvard.
Today I am going to talk
to you about
a huge unsolved problem
of how to visualize electrical activity
in the brain.
Now take a minute
and put your hand
on your head
and think about what you are touching.
Within a centimeter of your fingers
there is a piece of the most mysterious
and poorly understood matter
in the universe.
Here is a picture of your brain.
Now if we zoom in
by a factor of about 300
we see that this brain
is made up of neurons.
There is about a hundred billion of them,
and they are connected
to each other each by
about ten to the fourth,
10,000 connections.
If we zoom in again,
now by a factor of 10,000,
we see that each one
of these neurons is encased
in a lipid bilayer,
a little layer of grease,
which separates the inside of the cell
from the outside of the cell.
This bilayer is an electrical insulator
while the cytoplasm
and the extracellular medium
are conductors.
So you can think of it
a little bit like
a parallel plate capacitor.
Now there are many proteins
that sit in the membrane
and these proteins
can pass ions
across the membrane.
There are charge pumps,
which can generate a voltage,
and there are ion channels,
which can relieve this voltage.
These channels act
a little bit like resistors
except that they are non linear, so if
there is a change in the voltage,
they can have big changes
in the conductance
which can lead to these
electrical spikes
in the membrane voltage,
which we call
action potentials.
Every action potential
is about a tenth of a volt high
and lasts about
one one-thousandth of a second.
These are the currency
of the nervous system.
So every thought
and hope and dream
and feeling that we have
is encoded in these patterns
of electrical spikes
traveling through the
hundred billion neurons
in our brain.
That is all there is.
And so we'd really like
to see these action potentials
so that we can understand
what each neuron is doing
and also to look at
the global patterns of activity
and the interrelations
of the signals between
neurons to understand
how information
is being processed
within the nervous system.
So, basically this is a
voltage measurement problem.
Now traditionally this is measured,
as you might
measure the voltage
in any electrical circuit.
Electrophysiologists take a glass pipette
with a little electrode in it
and jam the pipette
into a cell and then there is
another electrode
out in the solution
and by hooking these up
to an amplifier
they can measure
the voltage difference.
And this works nicely,
but it only measures
one cell at a time.
It's extremely slow
and laborious to get these
electrodes into the cell,
and it doesn't give
you the global view of
what is going on in
a complex neural circuit.
Here is an artist's conception
of what we would really
like to do.
We'd like to just make these
neurons light up when they fire.
The problem is
that you can't see
the voltage in a neuron
anymore than you can see
the voltage in a telephone cord
when somebody is having
a telephone conversation.
We need some
contrast agent to convert
membrane voltage into light.
Now physicists and
chemists and biologists
have been working on trying
to invent contrast agent
since the late 1960s.
And this activity has really taken off
in the last few years.
Here are just a few
of the things
that people are working
on today. These are all from
within the last year or so.
People are trying to develop
voltage sensitive dyes.
So these are organic
small molecules which will
intercalate into the membrane,
and in some of these dyes,
illumination with light
generates an electronic excited state.
The charge distribution
in the excited state
is different than the
charge distribution in the ground state,
and so when there is a voltage
across the membrane
that voltage perturbs
the relative energy
of these two states
which can alter
the fluorescence properties
of the dye.
And so, this provides
a means to read out
membrane voltage with
fluorescence.
Other people are trying
to put semiconductor
nanocrystals
in lipid membranes.
These nanocrystals have
quantum-mechanical,
delocalized electronic states.
Membrane voltage can again
shift the relative energies
of these states
and adjust again the
fluorescent properties
of these crystals.
Other people are working
with objects called
nitrogen vacancies in diamonds.
These are tiny quantum
mechanical defects
in diamonds
and people think that if you
get a small enough
diamond nanocrystal
and you stick it into a membrane
the fluorescent properties
of these nitrogen vacancies
will again be sensitive
to membrane voltage.
Now I want to emphasize
these are all interesting
prospective technologies,
but none of these
have solved the problem
that I posed at the beginning yet.
In all of these technologies
the sensor is sitting
in the membrane.
And this is because
to measure a voltage difference,
you need to be connected
both to the inside of the cell
and the outside of the cell.
Other people, including myself,
are working on voltage indicators
based on fluorescent
transmembrane proteins.
The idea is to have a protein
which sits in a membrane
and to somehow
make its fluorescence
sensitive to the membrane voltage.
I'll tell you just a
little bit about one
indicator which we have been
developing in my lab.
The story starts here.
This is a picture of the Dead Sea.
There is a micro-organism that lives
in the water here that produces
a transmembrane protein
called archaerhodopsin-3.
This protein absorbs sunlight
and uses that energy
to pump a proton, a charge,
from inside the cell
to outside the cell.
This generates a membrane voltage,
which this creature uses
to power its metabolism.
So a few years ago
I said to myself
I wonder if we
can run this thing
in reverse.
Can we use a change
in membrane voltage
to induce a conformational
change in this protein
which will lead to a fluorescence
signal that we can see?
Just like this. Right.
And you see this works
in Powerpoint,
so that is very encouraging.
After several years
we engineered
a protein which
had these attributes.
And here is a picture
of a human tumor cell
where we have introduced
a gene for this protein
and we are using a little patch pipette,
which you can't see,
to induce steps of voltage
between plus and minus
a tenth of a volt,
and we are looking at the fluorescence
of the protein.
So here you can see
that as the voltage goes up,
the protein gets bright,
and as the voltage goes down
the protein gets dim.
So this is a
genetically encoded fluorescent
reporter of membrane voltage,
and it turns out
that it can be quite fast
and sensitive
but also very dim,
which makes it hard to measure.
We have now developed technology
to take these proteins
and to express them
in neurons in a Petri dish.
So the neurons become fluorescent
and their fluorescence
becomes sensitive
to the voltage
in the membrane.
We are also expressing
another protein in these cells
which renders them
sensitive to blue light.
This is a light gated
ion channel
and targeting a
flash of blue light
to the cell body
will trigger the
neuron to fire.
We have developed
computational techniques
to infer the propagation
of these electrical impulses
at an extremely high time resolution.
Here I will show you a movie
at a one hundred thousand
frames per second
of this optically induced
action potential
propagating through a neuron.
And you can really see
how these signals
propagate through a cell now,
but this is a neuron in culture.
We have taken a rat brain
and we've mushed it up
and we spread out
the neurons in a dish.
We'd really like to do this
in an intact brain,
but neither we
nor anybody else on the planet
knows how to do that right now.
There are several challenges to this,
and I'll tell you about them.
So one of the
problems we face
is that the brain
is not transparent.
Here is a picture
of a mouse brain.
And you can see
that it scatters light
very strongly.
It's about the size of my pinky
but light scatters
over a distance of
less than a tenth of a millimeter.
And so it is an
unsolved challenge how to
visualize what is going on
inside of this chunk of tissue.
We need to come up with new kinds
of optical or electrical tricks.
Another challenge
is that in every
mouse brain there is about
75 million neurons.
To record from these
at a thousand frames per second,
which you need to detect
action potentials
would lead to a
data rate of about
75 gigabytes a second.
We don't know how
to store this data, let alone
what to do with it
if we did have it.
So this brings me to
another unsolved challenge,
which is the interpretation problem.
So even if we had the best
technology
imaginable for visualizing
electrical activity in the brain,
how do we
interpret it?
We'd like to relate
the electrical activity
to some statement about
the underlying biology
of the cells.
What are the ions channels
that are active?
What's their state
of regulation?
What's their distribution?
What's the metabolic
state of the cell?
What's happening
to its energy supply?
We all experience that
as we go through our
day to night cycle
the function of our neurons
changes tremendously,
whether we are asleep or awake,
tired or not tired,
hungry or not hungry.
And those changes
are not mediated
by structural changes
in the brain but
by changes in presumably
the metabolic state
of our neurons.
And then finally we'd like
to infer from patterns of activity
how the neurons are connected
to each other.
To figure out who is calling
the shots and
who is controlling who
and how they
integrate and process information.
To illustrate this,
here is a
movie of some neurons.
This was a movie
taken in my lab.
These are neurons in a dish,
not in an intact brain
where we will look at
their firing patterns.
This is a movie at
about five hundred
frames per second.
The neurons have been
artificially colored
just so you can tell them apart.
And we can zoom in
on anyone of these cells.
and look at its firing pattern,
and we really don't
know how to interpret
these firing patterns
in terms of the ion channels,
the energetics,
and the connectivity of these cells.
So there is tremendous scope
over the coming years
to combine the efforts
of physicists, chemists, and biologists
to develop technology
which will let us visualize
the activity
in this little chunk of matter
that is right between
all of our ears.
Thanks.