My name is Daniel Colon Ramos.
I'm an associate professor in the department of Cell Biology and Neuroscience at Yale University.
I'm also an MBL fellow and an adjunct professor in the Instituto de Neurobiologia de
Universidad de Puerto Rico.
And today I'll be telling you about our work on the cell biology of the synapse and behavior
using the nematode C elegans, that you can see here.
Now, if you look at this nematode that we have lit up like a Christmas tree, and you
know how we did this... we... you know how we did this work, then you can skip on to
the second and third part of our lecture series, where we'll be talking about the specific
discoveries we've made using these transgenic nematodes.
If, however, you're looking at this and you're wondering, how is it that they can create
transgenic nematodes where they can see specific neurons and specific synapses?
This is the lecture for you.
By the end of the lecture today, you will know if... you will understand how we were
able to do this, and you will know, for example, how we image synapses in living animals.
I'll also explain how we're able to track behaviors and neuronal function in vivo,
and how we can use genetics to discover the molecular and cellular pathways that underpin the
cell biology of the synapse and behavior.
Before I tell you about how we do our work, I'd like to explain how I became interested
in these questions.
I think all scientists have a memory, something that they associate with becoming interested
in science, and this one is mine.
These are baby leatherback turtles that are hatching from a nest not far away from where
I was born and raised in Puerto Rico.
And I was always fascinated by the fact that these animals, from the moment that they are...
that they come out of their egg, they know exactly where to go.
They're bee-lining it towards the ocean.
And they didn't go to turtle school, so how is it that they know how to go towards the ocean?
And there's also something amazing that's happening here.
These animals, which will grow up to be about the size of a Volkswagen, about nine feet long,
they travel the world's oceans and when they have to make the most important decision
in their life, which for a turtle is where it's gonna lay its eggs, it will remember
this beach and it will come back to it.
So, with that, I'd like to underscore that animals' behaviors, like even these behaviors
that seem very complicated to us, when we think about them we can coarse-grain reduce them to,
for example, innate behaviors, or hardwired behaviors, and experiential memory.
In the case of these turtles, the innate behavior is the capacity of the animal to know that
it needs to go towards the beach when it's born, and the experiential memory is the capacity
to learn that it has to come back to that beach... which, depending what beach she was
born in, will change.
Now, they're both underpinning the architecture of the nervous system, the innate behaviors
in the developmental programs that lead to the formation of the circuit architecture
that underpins the animal's behavior, and the experiential memories in the
interplay between that circuit... circuit architecture and the experiences that the animal will have
throughout its life.
Now, if you're interested in these questions... these are very broad questions in neuroscience.
How is it that the architecture of the nervous system facilitates behaviors?
How... how can you make a question like that tractable?
How can you actually advance knowledge on a fundamental question like that?
And the way that we choose to do it in my lab is by using the nematode C elegans.
And the nematode C elegans is a tiny worm.
It's about the size of a comma in a sentence.
It's over here.
And, as I mentioned before, these... these turtles, the leatherbacks, they will remember
the beach... the beach in which they were born, they'll come back to lay its eggs,
like this one is doing here.
And C elegans also has behaviors that are modified by the experience of the animal.
For example, C elegans does not have an innate preferred temperature, like humans do.
C elegans, instead, can learn to prefer a temperature, depending on the conditions
in which it was raised.
Animals that were grown at 15 degrees Celsius, when putting a temperature gradient in the middle,
for example... these are worm tracks that you're seeing, here, and you... you can
know that they're all moving towards that side of the screen, which is the colder side
of the gradient.
Now, if you take isogenic animals, genetic... animals that are genetically identical to those,
and you now raise them at 25 degrees, and you put them in the same experimental conditions,
they will now move towards this side.
So, let me break this down.
The main difference between the animals over here and these animals here is not the genetics,
because they're isogenic.
It's not the conditions that they're seeing in the experiment, because they're the same.
It's actually the previous experience that they had.
And that previous experience altered the capacity of the animal to respond to these same conditions.
So, I'm interested in how that happens.
We have collaborated with a number of labs to establish experimental paradigms
that allow us to track these behaviors.
For example, we can take these worm tracks, we can align them with the same origin point,
like we're doing in this graph, and you can see them all moving, for example, when they're
raised at 15 degrees, towards the colder side of the gradient,
or towards the warmer side of the gradient, and we can quantify their behaviors.
But, importantly, we can use the nematode C elegans, which... which is a genetically
tractable model, as I'm going to be showing you in this lecture, to understand what is it
that's changing about the circuitry which enables the animals to respond to the
same stimuli differently.
Now, if... if you want to learn more about how other labs, besides my own lab, and how
historically the community has actually tackled the questions of behavioral genetics, I encourage
you to visit this website, where there are a number of videos.
This is actually a treasure trove from the MBL NS&B course where there are a number of
videos on this topic.
Regarding our own research, it turns out that the behavior I just showed you is controlled
by a discrete number of neurons that I have schematized over here.
And we know, for example, that this neuron that we are representing with this... with
this triangle is a single cell.
It's called AFD.
And it connects specifically to this other neuron, which is called AIY.
Now, the names are not important.
What's important is that we know the identity of these neurons, they're single cells, and
we know how they're connected to each other.
Because, for the nematode C elegans, we actually have a map of every single connection in the...
in the context of the animal.
And what my lab has done is that we have either adapted or developed single-cell promoters
-- which I'll be explaining in a second what they are --
to be able to do these type of experiments, here.
So, what we're doing here is that, in living animals... all these micrographs were taken
in live animals.
We are labeling single cells.
So, there are hundreds of other cells that you're not seeing in this picture, because
we have labeled specifically the AFD neuron, which looks like this.
This is the cell body, here.
This is the axon.
And you can see the dendrite over here.
Now, we have done that for the sensory cell, which is AFD.
We have also known it for the downstream interneurons.
And the important concept here is that we can label, in the context of the living animal,
single cells.
So, we have the animal behaving and we can look at the cell morphology.
So, how can we do this?
How can we label single cells in living animals?
And that's what I'll be explaining in the next few slides.
They way that we do it is by creating what are called transgenic nematodes.
These are nematodes that carry genes that are normally not present in other sibling
nematodes in the wild.
These are nematodes that only exist in the lab.
And this is how we do it.
We actually have... this is the body of the nematode, here.
This is the gonad that you're seeing here.
And this... this little thing here is the... it's a... it's a needle that's gonna go into
the animal as you're gonna see in a second, and we're injecting the gonad... boom.
There.
We injected it with DNA.
So, that's the technical part.
If you were a technician, that's what you do.
And then, in the next generation, you collect animals that are transgenic.
But what is it that we are injecting there?
That's the important question.
And I'll show you in a... in a few seconds, essentially, we're injecting DNA that carries
the instructions that are gonna label these specific neurons.
And this DNA, which... which is called a transgene, it conceptually has three parts that are important.
So, if you understand these three parts, you'll conceptually understand what this is about.
It has a promoter and the promoter is essentially a genomic region, a part of the DNA,
that drives expression in specific tissues.
So, this is a part of the DNA that's usually upstream of a gene and it tells the organism
where that gene should be expressed.
If you understand where the promoter drives expression normally, you can use that
same information to then drive expression of whatever you want in that cell.
So, it's the promoter.
Then, it's the cDNA.
The cDNA is the coding DNA, cDNA.
And the cDNA essentially has the instructions that will encode a given protein or a gene.
And then we have a probe.
And the probe can change, but it's... it's usually something that you are...
that you use to visualize a biological process.
So, a common probe that we use is GFP, which is green fluorescent protein.
And I'm... in the next five or ten slides, I'll be going one-by-one and explaining
each of these different component systems.
So, let's start with the promoter.
The promoter, as I mentioned, is a genomic region and what happens in an... in an organism
like C elegans is that there's a community of researchers that every time they discover
a gene, they characterize where that gene gets expressed.
So, you know not only the gene that you discovered, but you also know which tissues that gene
gets expressed in.
And using that knowledge... there were a number of genes that were discovered by other groups
that were expressed cell-specifically, for example, in the AFD neuron.
So, because we knew the identity of a given gene that gets expressed in the AFD neuron,
we can look at that promoter.
And we're not particularly interested in that gene, but we can use that knowledge, we can
use the promoter region, and then drive the expression of whatever we want in that neuron.
Because those... those instructions are modular.
You can... it's kind of like a little module that you can take away from that gene and
use it to drive expression of something else.
In this case, we're using the cell-specific promoter of AFD to drive GFP and that's how
we're able to image, in the context of the living animal, this specific cell.
We can do the same thing for AIY and for the downstream interneurons, and that allows us
to probe the morphology of these neurons in the context of the living animal.
So, that's how we do that part.
But what is... what is GFP?
What is it that we are driving that allows us to see these cells?
Before I...
I give a brief introduction of GFP, I'd like to mention that there are a number of excellent
lectures that go more in depth.
Actually, the Discovery lectures by the people that made the discoveries that I'm about to
describe in the iBio website, so I encourage you to go and visit their website and...
and learn more about this.
But, very briefly, GFP is a protein that is normally expressed in the wild by this beautiful
organism, which is a jellyfish.
And it expresses, essentially, two proteins that allows it... that allow it to glow in
the dark.
One of them is aequorin, which is a molecule that reacts to calcium and emits blue light.
And then there's a second protein called GFP, or green fluorescent protein,
that then receives that blue light and then shines, click, green.
And what scientists did is that they identified what those genes were.
The scientists that did that are actually over here.
They won the Nobel Prize for their discoveries because this was a... essentially a curiosity-driven
fundamental discovery that changed the way in which we can image proteins or image cells
in living organisms.
The person that originally identified the proteins that the jellyfish is using to shine light
was Osamu Shimomura and... it's this scientist over here.
And then Martin Chalfie was the first person that put them into an organism, actually,
he put them into C elegans.
And Roger Tsien, he was the person that developed a number of different colors that enabled
more versatility and use of these probes.
So, again, Osamu Shimomura, what he did is that he took the jellyfish and this...
I want to emphasize this, because it's very important to understand how scientific discoveries
are made.
The first point is that this is curiosity-driven research.
So, Osamu Shimomura did not start with a question of, like, let's try to cure this disease or
that disease.
He started just by wanting to understand how is it that this jellyfish is able to grow.
But, because knowledge begets knowledge, and it's cumulative and scientists kind of
build on each other's discoveries, we were able to use those very important observations to...
to revolutionize the way that cell biology is done.
So, he purified the proteins.
Then, there was another scientist that actually did not receive the Nobel Prize, but what...
is recognized by the community because his... his contributions were fundamental to
the development of GFP as a probe.
And that person is Douglas Prasher.
And essentially what he did is that he... once the protein was identified, he identified
what gene encoded that protein.
He was very collaborative and generous with his knowledge, and that allowed Martin Chalfie
to take that gene and put it... with the promoters that I explained earlier, put it into different
tissues in C elegans, and be able to observe the morphology of these tissues, much like
I explained in the first part of the talk.
And finally, the... the original protein that the jellyfish makes is a green fluorescent protein,
but that protein can be altered to glow in different colors.
And that was the work of Roger Tsien, which increased the versatility of probes that can
be used in vivo, and simultaneously, like you're seeing here in these tissues,
to be able to image different cells.
And using those type of probes, you can do things like... you can have, for example,
green fluorescent protein, yellow fluorescent protein, red fluorescent protein.
These are actually bacteria that are expressing different fluorescent proteins.
And they can... this is... this is from Roger Tsien's lab, and you can draw beautiful scenes
with them.
But, importantly, for our work, you can label single neurons with these fluorescent proteins.
And again, I want to emphasize that if you want to learn more about this you can go to
the iBiology Seminar Series and the Discovery Series
to hear more about how these discoveries were made.
Now, this is how we can label and... and create an animal like this.
This is an image that was gifted to me by Marc Hammarlund,
my friend and colleague from Yale University,
and we are labeling, here, GABA neurons and acetylcholine neurons,
which are two different neuron classes.
And you can actually distinguish them.
Some of... some of these neurons are right on top of each other, so you see them in yellow,
but others are in the head region and you see them in red, or you can see these... these
neurites that are crossing, in green, and they correspond to different neuron classes.
And we can do this, in part, because the nematode is transparent.
So, what do we use that for in my lab?
So, essentially, as I mentioned, we're interested in understanding how the architecture of the
nervous system facilitates behavior.
And I will argue that keystone, central to that architecture, are synapses.
So, synapses are points of connectivity between cells, like you're seeing right here.
And since we've recognized... and by we, I mean the field... recognized that neurons
are specific cells, we've known that see... where the positions of the synapses are specified
is critical for specifying the function of the circuit.
Because when, where, and how a synapse is assembled and modified establishes... establishes
how the information is gonna flow through the circuit.
So, we're interested in that cell biology of the synapse.
And we're interested in the cell biology at two levels.
One of them is, how is it that synapses are built and maintained to be able to sustain
the architecture of the nervous system?
So, how... how are they established during development to be able to sustain behavior?
And the second one is, how is it that they're modified, by the experience of the animal,
to... so that the animals can learn?
And there's an inherent tension between these two points, because what makes you really
good at... in this point, here, which is essentially the stereotypicity, the rigidity of...
of positioning synapses will make you weak over here, which is the plasticity, the flexibility.
So, we're interested in how the cell biology of the synapse resolves that tension.
And to exemplify our work, I'm gonna use a quote by the person that essentially discovered
the synapse, which was a Spanish scientist called Santiago Ramon y Cajal, who first described
the synapse as...
I'm gonna read it in Spanish and then in English...
"Osculos protoplasmicos que parecen constituir el extasis final
de una epica historia de amor",
which translates into,
"Protoplasmic kisses that appear to constitute
the final ecstasy of an epic love story."
This is the first description of the synapse.
So, who... who doesn't want to work on that?
Come on.
So, essentially, I...
I love the metaphor, but what I like particularly is that you can think of the synapse as
the canary in the mine.
If you're able to visualize the synapse and you can alter that synapse in some way,
you can figure out what is important for the formation of that synapse, you can figure out
all of the steps that need to happen in the plot.
That plot that leads to these two neurons finding each other and kissing... that final,
like, kiss at the end of the plot.
If you can disrupt that, you can figure out what are the important parts of the plot.
And that's what my lab does.
So, how do we do that?
So, we look at the cell biology of the synapse in development and behavior.
And we do that by, again, using this concept but, over here, we... for the cDNA, we actually
use synaptic proteins.
So, let me explain how we use synaptic proteins to be able to probe the... the plot of the
neural development as the synapses are being formed in the animal.
So, I showed you this image earlier.
So, this is just expressing the... the probe cytoplasmically, so it's all over the cell,
so you can look... it's very useful because you can look at the cell morphology.
But if you want to see where the specific synapses are, first you need to understand
which proteins are actually localized at the synapse.
And it turns out that a synapse... it... it's formed of two parts: the presynaptic neuron,
which talks to the postsynaptic neuron.
And here we have it uhh... schematized, here.
Presynaptic talking to the postsynaptic.
And importantly, the presynaptic region is formed by synaptic vesicles.
These are like little balloons chock-full of neurochemical information.
So, that's what the neurons use to communicate with each other.
And those vesicles are gonna fuse with this area that I... that I represent with this...
this dark region.
And that region repre... what it... what it constitutes is just... think of... think of
it like a... a loading dock.
It's an area where the vesicles fuse and then they release the neurotransmitter information
into the postsynaptic cell.
But both the vesicles and this region, which is called the active zone, there are a
number of proteins that form part of it,
and what my lab has done is that we have used that
knowledge that has been generated by others to label those proteins and visualize
where the synapses are.
So, here, for example, we have a synaptic vesicle associated protein called RAB-3 and
it's labeled with GFP.
So, it's fused to GFP.
And you can see that the... the pattern of this image is very different from the pattern
of this image.
This is the same cell.
But you're seeing a different... a different region is being labeled.
Here, for reference, I put a dashed box in this same area of the cell.
And you can be... you can see that this region here, for example, it's not present in the...
in the darker image over there.
And the reason is because there are no synapses here.
So, when you look over here, you don't see any synapses either... or you don't see synapses
in the cell body, but you do see synapses in this region, here, which is...
corresponds over there to that... to that dashed box.
So, we can look at the distribution using these markers of the... of the synapses along
the neurite uhh... by labeling the synaptic vesicles.
Or, also, labeling the active zone.
So, here's another image.
And you can see these two images are actually very similar to each other.
Now, they correspond to two different animals
and they correspond to two different subcellular structures.
So, that just tells us that... that the synaptic distribution is... it's actually stereotyped
across animals.
And that's important for us as geneticists, because what we do is that we use this pattern
and we try to break it, to find what molecules are important for the formation of this pattern,
as I'll be explaining in the next couple of slides.
So, we can... we can image these synaptic vesicles.
We have... we have also developed probes, not only to image the presynaptic sites,
as we're doing here with the vesicles and the active zones,
but also over a dozen different cell biological markers
that label the mitochondria and other organelles, and we can label any
of those organelles in any of these cells.
So, we can look at the cell biology of the synapse in the context of the living animal.
And then we have collaborated with a number of people, including my friend and colleague,
Hari Shroff at the NIH,
to be able to develop microscopes like the one I'm showing here,
which is the dual-view selective plane illumination microscope that Harry created to... to observe
how the nervous system forms in the embryo.
So, here's a movie of the type of images that we can generate.
Here, we're labeling all neurons and you can see the outgrowth of neurites in the embryo,
which is right here.
And you can see a ring over here, which corresponds to the nerve ring.
It's gonna become very clear in this image.
That's the brain of the animal.
So, we can see the formation of the brain of the animal in the living organism.
We can also label specific synapses.
If you're interested in how this microscope works and how it enables us to be able to
image these processes with the probes we've developed in the early embryos, I encourage you
to look at Harry's seminar on how light sheet microscopy works.
And, also, I have a seminar where I talk more broadly about collaborations and how they're
so enabling in science.
So, the collaborations are a big part of our... of our research program.
But, for now, I'd just like to say that we can use these microscopes in collaboration
with a number of groups, including Zhirong Bao's group in Sloan Kettering
and Bill Mohler at UConn,
to be able to understand, how is it that the nervous system comes together
in the early embryo?
So, this is a consortium that is building what will be a movie, for the first time in
any animal, of all of the decisions made in neurons as they're coming together.
Now, uhh... the way that we can do that, as I was mentioning, is through the visualization
of specific proteins that we have tagged with GFP.
So, this is this category here.
I'd like to tell you a little bit about this final category, here, where we can use the
promoters that... that have been identified, both with GFP but also with other sensors,
or ways of killing cells, to be able to then link the cell biology with the behavior of
the animal.
So, I mentioned earlier that we can do these behavioral assays.
This is how wild-type animals look.
Just a reminder, if they're grown in the cold they will move in this direction;
if they're grown in the warmth they will move in this direction.
So, we can take animals and we can express, in a population of animals, using these cell-specific
promoters that I... that I have been discussing, we can express caspases or other
proteins that kill these cells in individual animals, but in a population.
And then we can do experiments where we can determine, when we kill that neuron that...
that you're seeing there, AFD, the sensory neuron, these animals are now incapable of
performing the thermotaxis behavior.
So, that tells us that that neuron is very important.
And, if we want to understand what that neuron is doing, what it's responding to,
we have developed rigs or equipment,
in collaboration with a number of labs, including Aravi Samuel at Harvard University.
Here, this is a microscope with a little piece of equipment that allows us to very precisely
change the temperature and then visualize what this neuron is doing.
And we can visualize what that neuron is doing by using a probe that I'll explain now,
which is called GCAMP6.
GCAMP6 is a probe that is a calcium sensor, so it allows us to see changes in calcium
in the neuron.
And with this rig we can give a temperature stimuli, like you're seeing here, and that...
that... what you're gonna be seeing is that... essentially, we're changing the temperature
very finely.
That red line represents the temperature at which the animal was raised.
And you're gonna see that... when I move this placeholder, you see the cell body and you're
not gonna see anything when it's below the threshold, or the temperature where it was raised,
but now it's like a sing-along.
Every time that it goes above the temperature... uhh... above this threshold, every time that
it goes up, it... this... this neuron will fire, or you'll see calcium transients.
So, that tells us that this neuron is responding to increases of temperature,
above a given threshold.
And I'll be talking more about this work in the third part of the... of the... of the
lectures.
And that's essentially how we can do this work.
This... this is how we can link the cell biology of the synapse with behavior and understand,
how is it that the synapses are established during development?
And how is it that they're modified with behavior?
Now, we can... we can probe these phenotypes, we can probe the wild-type behavior of the
animal, the wild-type distribution of the synapses,
and the morphology of the neuron in the context of the living, behaving animal.
But how can we disrupt it to identify the genes that are important for the normal functioning
of the nervous system of the animal?
And that's where genetics comes in.
And I'll give a very brief primer on genetics to explain how is it that... that we do our
work and make our discoveries.
So, very briefly, when you think about genetics, you can think...
people talk about forward genetics and reverse genetics,
which are two terms that are kind of confusing if you're
not in the genetics field.
But I'll break it down conceptually in this slide.
So, forward genetics... essentially you start with a biological process.
So, in our case we can start with the behavior or we can start with the position...
the normal distribution of the synapses.
And then what you're doing is reverse engineering.
So, you know, when you have a little kid that wants to understand how is it that a toy works
and they break it to see what the important parts are?
That's what we do.
So, we have a biological process that works well and we break it.
And I'll explain how we break it in a second.
Once you break it, you isolate ones... animals, essentially, that are mutants, meaning that
they... they're not performing the process normally.
You identify what you broke, which is important, and then you analyze how is... how it is important.
So, that's forward genetics.
This is what most people associate with genetics.
There's also reverse genetics, where you actually start with a gene.
So, you start by... by having a candidate of something that... that...
that you suspect will be important,
and then you have a biological process that you analyze to see
if that specific gene, that you hypothesize,
is important for a given process,
if it's actually important for the given process.
So, in order for... to... for... for anybody to be able to do with genetics,
including us, we have... you need a phenotype.
In our case, our phenotypes are the behavior of the animal that I showed you,
the thermotaxis behavior, or the synaptic positions.
Then, you mutagenize a bunch of worms.
Now, this is... people get confused about this.
Here's the main concept.
You are randomly putting... you're putting a chemical.
In... in our case, we use EMS, which is a chemical that's frequently used to
mutagenize animals.
And you're creating a bunch of different mutations.
So, yes, you're gonna have animals that are gonna die, and they're gonna look weird in
a number of different ways, but because you have... your... the phenotype of interest,
you only pick up mutants in your phenotype of interest.
You're not picking every mutant under the sun, because then you're gonna have a
ton of mutants and... and... and too little time to characterize all of them.
So, we have the synaptic position -- we pick up mutants that affect the synaptic position,
for example.
So, it's unbiased.
You don't know what you're affecting.
You pick up the ones with interesting phenotypes, or the abnormal ones.
And then you figure out where the genetic lesion that resulted in that phenotype is,
which I'll be explaining in a slide.
The... the main concept of how is it that we can figure out where the genetic lesion
is once we have a phenotype is explained very briefly here.
And for the purpose of today's explanation,
I will color the two DNAs in these two different colors.
And essentially, the main concept here is that we can tell the difference between the
DNA that comes from this strain, which is our mutant strain, and the one that we use
to map, which is a special strain.
In our case, we use one that's called Hawaiian.
It's a strain of C elegans that was isolated in Hawaii.
But it can be a number of different mapping strains.
The... the important concept, and this is the most important part, is what I'm representing
in color, here, are what are called single nucleotide polymorphisms.
And don't get confused with the lingo.
It just means that we can differentiate the DNA
that's coming from each of these two original strains.
So, here we have our mutant, that looks like a little fat worm, and we're mating it with
the Hawaiian one, which I'm representing here with this beautiful flower color.
So, we... we made them and then, in the offspring you're gonna have animals that are gonna be
heterozygotes, so you're not gonna be able to see the mutant phenotype.
Here, I'm assuming the mutant phenotype is a recessive single genetic lesion,
for the purposes of today's explanation.
So, you don't see it, because it's a recessive lesion in the context of the wild-type animal.
Then, you self those animals, and in the next generation you're gonna have a bunch of animals
that don't see the phenotype.
Now, here, you have to understand, we're tracking the phenotype, because we don't know what
the... the genetic lesion is.
We're tracking the phenotype to find the genetic lesion.
So, we don't know if they look like this, but we do know that they look like that.
That we know that they don't look like fat and little.
So... so, we hypothesize that we have a heterozygote animal.
And then... we have animals, however, that are... that have the mutant phenotype of
the original parent strain.
And they're gonna have... they're all gonna have the lesion in the same part of the...
of the chromosome.
And the lesion has to come, and this is the most important part, it has to come from this
orange DNA, so to speak.
It has to come from this original strain.
And it has to come from there, because that's where the mutation came from.
So, now you just have to identify the areas of the DNA in the offspring that look mutant,
that have that orange DNA.
That's essentially what you're doing.
So, for example, if you have this animal, you know that the lesion cannot be in
this region, because it has red DNA, so it cannot be there.
In this animal, it cannot be in this region because it has red DNA.
And in this animal, here, it cannot be in this region because it has red DNA.
So then, you kind of narrow it down to the region.
And you do this iteratively, many times, until you identify the genetic lesion area.
Now, the... the... the main concept of the mapping is that you have two strains.
The mutation needs to be in the original strain, it cannot come from the red strain because
this red strain didn't have the mutation, and you can tell the difference between the
two strains by what's called DNA fingerprinting, which is essentially showing differences between
the two DNAs, which I'm representing here in color.
If you understand this conceptually, you understand genetic mapping.
So, the work that we have done, for example, and I'll go into more detail in the
second part of the presentation, but just to give you an example of how we used these techniques,
all together, for discovery is that we can start with a phenotype,
this is the wild-type phenotype, this is the distribution of the synapses.
We do a forward genetic screen, we identify a mutant in which that distribution is abnormal.
Hopefully you can tell the difference between that picture and the one that is right next
to it.
So, it's a mutant phenotype.
And then we identify, what is it that we affected here that affects the distribution of the synapses?
So, it turns out, by... by doing that kind of analysis and then identifying where these
genes are acting to be able to establish the correct umm... development of the synapses,
we discovered that there's a gene called Netrin, which is expressed by glial cells, and that
that gene is essentially specifying where the presynaptic sites that you're seeing there
in the... in the wild-type image, where they're gonna form.
And it does so by instructing the localization of its receptor, which then drives
a signal transduction cascade that ultimately culminates
in the organization of the cytoskeleton and the clustering of synaptic vesicles.
So, the reason that, for example, in this mutant, here, you have fewer synaptic vesicles
in this area as compared to the wild-type animal over here, is because you have a mutation
in... in the instructions that are specifying the form... the... the clustering of the vesicles, there.
And if you alter that in single cells, then you can also alter the behavior of the animal,
which we'll be talking about in the part two of the presentation, but the main concept
that falls out of these type of discoveries, the main contribution of this type of work,
is the fact that, by doing this type of work, we... we established that glia specifies,
in living animals, the site of synaptic formation.
And this was surprising because we were hypothesizing that what was gonna be specifying the site of the
synaptic connectivity will be the postsynaptic cell that the...
that this neuron is talking to.
And it turns out that it's not the postsynaptic cell.
It's actually kind of like a matchmaker cell, which is called the glia, which is telling
those two cells to meet at a... at a specific place and connect to each other.
So, hopefully this example brings together all the concepts that I've been describing.
But the... the process of discovery, both for our labs and other labs in... in... in
C elegans, essentially what it does is that it allows you, in this simple organism,
to link a number of fields that are usually disconnected in other fields,
like genetics, development, cell biology, physiology, imaging, etc,
to be able to make gene discoveries of... of
fundamental processes that control neural connectivity.
So, I hope this lecture helped illustrate the three points that I'm making here.
How is it that my lab is able to image synapses in living animals.
How we can track the behaviors and then link the cell biology of the synapse with the behavior
of the animal.
And how we can use genetics to uncover new mechanisms that underpin the cell biology
of the synapse and how it regulates the behavior of the organism.
In the next two talks, I'll be taking these concepts that I explained today, the techniques,
and I'll be giving you specific examples of discoveries that we've made using them.
So, in the... in part two of this series, I'll be talking about, how is it that synapses
are built and maintained in the living organism?
And in the part three, I'll be telling you about how synapses can be modified to accommodate
learned behavioral preferences.
Underpinning these two interests is our aspiration to understand the cell biology of the synapse,
both in development and in behavior.
With that, I'd like to thank the people of my lab that did the work, and also the
funding agencies that made our work possible.
Thank you.
