In the last video,
we set an important goal for modern neuroscience:
to determine the causal mechanisms
through which brain function drives mammalian behavior.
We thought that there were
three key aspects that needed to be done.
We need to measure neuronal activity
and the synaptic connections between neurons during behavior.
We need to be able to manipulate those neurons
and monitor the impact upon other neurons in the brain
and also, of course, see what impact that has upon behavior.
And if we can combine that measurement and manipulation
of neuronal activity with sufficient resolution in time and space,
then it would be important to quantitatively see
to what extent we've really understood how event A causes event B
that then finally causes the animal to move,
driving a behavior.
So how are we going to do that?
Many of us, of course,
dream of understanding the thought processes of the human brain.
We have obvious questions: How do we perceive the world?
How do we remember?
How do we do the things we like to do?
And of course, we're also interested
in curing the many brain diseases that affect large populations.
Now, it's likely that it'll take a long time
before we understand the details of how the human brain works.
On the other hand, neuroscientists have high hopes
that we'll get a detailed causal and mechanistic understanding
of how the mouse brain works,
perhaps in our lifetimes, and likely within the 21st century.
So let's have a look and see how close the mouse brain is
to the human brain, and how likely it is
that we'll be able to learn from the mouse and translate that into anything
that relates to what goes on in the human brain.
A good starting point, in terms of thinking about man
in the context of other animals
is to think about evolution.
So here we have man that diverged from its common ancestor
with the apes, the chimpanzee, for example,
some tens of millions of years ago.
If we go back some further tens of millions of years ago,
we diverge from the monkeys,
and here we have within the primates, of course,
our closest relatives,
and of course, it's extremely interesting to understand
the behavior of monkeys and apes
and of course, in the context of their brain activity.
But, of course, detailed mechanistic investigations
into brain function in monkeys and apes
is extremely difficult to do.
The mouse, on the other hand, diverged some 80 million years ago from man.
That's when we had our last common ancestor,
and within at least the context of evolution,
mouse and man are close to each other
compared, for example, to birds and reptiles
where the mammals diverge from the rest of the vertebrates
some 300 million years ago.
So on an evolutionary timescale,
mouse and man are relatively close to each other.
If we look in terms of the genes that code for the various proteins
that carry out functional roles inside our cells,
about 99 percent of the genes that are found in man
have homologues in mouse.
So if there's a given genetic mutation in man
that's linked to some disease,
there's a 99 percent chance that we can find
a homologous protein in a mouse
and because of its strong homology in terms of the genetic sequence,
it's likely that it's going to be carrying out a related function.
So at least from a genetic perspective,
mouse and man are very close to each other
and we may be able to learn a lot at that level of detail
comparing mouse and man.
The mouse brain is, of course, different from the human brain.
The most obvious difference is in its size.
So very much like the overall size of the mouse
is much smaller than man's.
So mouse typically has a weight of around 30 grams,
and a human might have a weight of around 60 kg
so there's about three orders of magnitude
in terms of the difference in their size
of man and mouse.
There's also about three orders of magnitude difference
in the size of brain,
and so the human brain is about 1.5 kg in weight,
has some ten to the power of 11 neurons,
whereas the mouse brain is much smaller.
It's about one centimeter in length dimensions,
weighs about one gram,
and has some ten to the eighth neurons present in it.
So there's about three orders of magnitude difference
comparing mouse and man brains,
and in the midst of this is the monkey brain
that's also used in neurophysiological experiments,
and of course, is much closer to the human brain
in both size and number of neurons.
So there are big differences in the size of the mouse brain,
and that scales more or less with the size of the body of the animals.
Now in the same way that there's a strong homology
in terms of the overall layout of the human body and the mouse body,
we have four limbs, we have lungs, heart, kidneys, livers,
the internal organs are all the same.
There's also strong similarities in terms of the organizing principles
of the human brain compared to the mouse brain.
So if we now expand the mouse brain
and make it look like it's the same size as the human brain,
we'll see that many of the parts are the same.
So here we have the spinal cord and of course the human brain sits
here on top of the spinal cord also.
We have the brain stem and the brain stem,
we have the cerebellum of the mouse and the cerebellum of man,
and here with a large cerebral hemisphere of human,
that also is there in the mouse in the form of a large neural cortex.
If we now look at the different subdivisions of the neural cortex,
we also see that the organization there is strikingly similar.
At the posterior pole of the human brain
we have the visual cortex,
and that's also where the visual cortex of the mouse is.
Our feeling of touch, somatosensation,
is located around here in the human brain,
and it's also somewhere centrally placed here in the mouse brain.
Hearing, the auditory cortex is located somewhere around here,
in similarly displaced locations in mouse and man,
and the motor areas of the human and the mouse are also similar,
with their motor areas being in front of the somatosensory areas in both cases.
The human brain has a vastly expanded prefrontal cortex
that's relatively small in the mouse.
On the other hand, the mouse has a relatively large olfactory bulb
that's only much smaller in the man, and only just drawn in here, sketched
in this particular diagram now by me.
So the overall organization of the brain is extremely similar,
even at the level of where different brain areas
are processing different forms of sensory information.
If we look at the subcortical areas.
So here we have the outer layers of the mouse brain,
the neural cortex, and that's also how it is in the man.
The outer layers forming the neural cortex.
Below that we have the striatum, we have the thalamus in the mouse,
and similarly in man we have the striatum and we have the thalamus located here
and there are many other subcortical nuclei
that are organized in similar locations,
express similar genes, have the same receptors,
and apparently, play highly homologous roles
comparing mouse and man.
So the large scale organization of man and mouse brain is very similar.
But even if we look at the details,
if we say, zoom in on a patch of neural cortex,
we find that there are very similar features.
So there are different layers, so the mouse brain is divided
into layers that are very obviously different from each other,
and you see the same layers in the human brain
and the cells that are present there have a very similar structure.
So there are large pyramidal cells in layer five
in both mouse and man,
with big apical dendrites and big dendrites here,
tufts, in layer one.
Equally, there's GABAergic interneurons present in both man and mouse
and there are strong similarities in the organization then,
both of cells and at the large scale
comparing man and mouse.
So by studying the mouse brain,
it's likely that we'll learn a lot about how the human brain is organized.
Studying the mouse has been greatly helped by a number of large-scale projects
that have been undertaken.
The Mouse Genome Sequencing Consortium
published the mouse genome in 2002
and the entire mouse genome is freely available
on a variety of internet websites.
And so here from the NCBI,
the National Center for Biotechnology Information,
you can, for example, search for your favorite gene,
perhaps the AMPA Type II receptor
is what I've searched for here,
and if you do that search you find out that this glutamate receptor
is located on chromosome three of the mouse genome.
You can click on this and you get the entire DNA sequence
for the coding region, as well as the surrounding introns
that then gives the promoter sequences for this gene
and determine where it's expressed in the mouse brain.
So we've learned a lot about the genome sequence of the mouse,
and of course, molecular biologists are being able to use that sequence
in order to introduce mutations and transgenes into the mouse genome,
and so, we can also change that mouse genome.
So, for example, if we know of a mutation in a human
that's linked to a genetic disease,
we can insert that mutation into the mouse genome
and study the effects of it.
There are of course, many different mouse mutants
that have been made for many different purposes
to try and help the advances in medical research.
Typically, when investigators make mutant mice,
they then donate those mice to different repositories
which then serve as mouse libraries, and one of the most important ones
is the Jackson Laboratory in America, Jax,
and if you go to this website you'll be able to see a complete list
of the mice that they have.
You can search by different genetic mutations
or insertions that have been made, and these mice are then a good way
in which different researchers can share their resources.
So typically when a mutant mouse is made, it's deposited here,
and then other investigators can take those mutant mice
and investigate them for their own particular purposes.
So that's a way in which resources are being shared
amongst mouse investigators
and that then helps advance research dramatically.
One of the key ways in which we can differentiate different parts of the brain
is through gene expression.
So in early development as the fertilized egg divides
and gradually forms the different specializations of the body,
our different organs, our different tissues,
that is accompanied by differential gene expression
so that's one of the things that differentiates a cell
in the kidney from muscle, from brain, is that they express different proteins.
They need to do different jobs, and so they express different proteins.
And within the brain, there are many different cell types
and in part, they can be differentiated
through their expression of different proteins
and an important advance and tool for investigators
looking at the mouse brain has been the Gene Expression Atlas
carried out through the Allen Brain Institute
and again, freely available on the internet.
You can go to this address, you can search for your favorite gene,
and you will then see throughout the entire mouse brain
where that gene is expressed.
So here I've searched for the dopamine type I receptor gene
it's stained here through in-situ hyperization
of saggital sections of the mouse brain.
I'm showing you one particular section here,
and you'll see that these black dots here correspond to individual cells
that express the dopamine I receptor.
That turns out to be largely restricted to the striatum,
something that we already discussed last week
with the GABAergic projection neurons
located here in the striatum.
This, of course, is just one section of the brain.
You can look at many other sections
and they have the entire 3D structure reconstructed quantitatively
in terms of the density of the in-situ hyperdization signal
and if you go to the internet you'll be able to spin this around
and get a very good feel
for the expression pattern of this particular gene.
That's, of course, extremely important in terms of understanding the anatomy
of the mouse brain, and in terms of thinking about interventions,
treatments and molecules that will affect brain function.
It's extremely important to know
what different proteins are actually expressed,
so this Mouse Gene Expression Atlas is serving an extremely important role
for neuroscience research.
The expression of different proteins and different cell types of the brain
is clearly extremely important in order to understand the function
of these different cell types.
So one thing that we can do through mouse genetics
is to label these different cells
through expressing different types of proteins.
So for example, we can take green fluorescent protein
and put it next to the dopamine Type I receptor in the mouse genome
and then the cells that express green fluorescent protein
are the same ones that express the Type I dopamine receptor.
So we can then cut brain sections and study their electrophysiology
and the synaptic inputs onto these D1 expressing neurons.
That's an extremely important thing in terms of understanding the role
of different cell types in the mouse brain.
Even more interesting is the possibility to express other proteins
that might be functional reporters, or perhaps enzymes
that might act upon other genetic elements,
and a great deal of advances have been made
through the expression of recombinases
that combine different DNA elements and then you can make
different genetic manipulations in a highly targeted way.
So I'll briefly describe the so-called Cre-LoxP system
that has become extremely powerful for making precise genetic manipulations
in the mouse genome.
This is something that was first developed from bacteriophage P1
but it doesn't have an endogenous counterpart
in the mammalian genome.
So we can exogenously insert LoxP sites
which are simply DNA sequences of 34 nucleotides long,
highly specific sequences,
you can insert those into, say, intron sequences
that surround a genome.
Intra-serve, this is the genome,
and we have a gene of interest located here.
We can, in intron regions, put LoxP sites surrounding that gene,
so that's simply inserting these 34 nucleotide sequences
in surrounding elements around where the gene is normally expressed,
and doing that, hopefully wouldn't affect the expression pattern of that gene.
So the mouse by itself would be wild type
and the only thing we've done is to introduce these small genetic elements
into regions that flank our gene of interest.
Now, what Cre-recombinase will do
is to recombine between these two LoxP sites
and then remove the gene of interest, creating a knockout.
We can do that in specific cells because we can express Cre-recombinase
under promoters that express in specific cell types,
for example, like in the D1 receptor expressing cells.
So we can cross one mouse that has these so-called floxed genes
flanked by LoxP sites, we can cross that by another mouse
that expresses Cre-recombinase in specific cell types,
and in those cell types, but not in the other cell types of that mouse,
we'll then create a situation where the LoxP sites recombine
and the gene sitting in between those is removed
and degraded and knocked out.
So we can make very precise, highly specific genetic manipulations
in well-defined cell types,
and that's something that's really essential
for causal and mechanistic understanding of brain function.
One of the interesting things is that the Cre-LoxP system
isn't the only one, but rather there are many.
So for example this Flp-FRT system which is very similar,
but acting on different genetic elements and one can then combine
the different types of genetic manipulations
to get that combinatorial specificity
in which one can make multiple independent genetic manipulations,
perhaps even in different cell types.
In addition to these interesting advances in the genome manipulation of the mouse,
there's also increasing data relating to the connectivity
of different brain areas inside the mouse.
Again, the Allen Brain Atlas has take the lead here
in providing online data
for where different cells project in the mouse brain.
Here I'm showing you one particular example,
where they're looking at the injection site here
in the primary somatosensory cortex
and injecting their tracer, they then see
where axons from neurons located in this particular area of the brain,
where they project to, in terms of other parts of the brain.
They've made injections of many different parts of the mouse brain.
Each one of these dots here corresponds to an injection site
from where they're mapping where the axons go from that brain area.
And I've selected the primary somatosensory cortex,
that barrel field, the whisker representation
that we've already thought a bit about in previous weeks.
So here's what the data looks like.
They've made an injection of their tracer
into this area here, the primary somatosensory cortex of the mouse,
and in this cornal section of the mouse, you see that the axons go to the striatum,
they go to the thalamus,
they go to the contralateral somatosensory cortex,
and if we now look at some of the whole brain view,
and again, if you go to the website
you'll be able to spin this around in three dimensions
and get really quite a good impression of where these axons are going,
you'll see that the primary somatosensory cortex
projects to motor cortex, contralateral cortex,
striatum, thalamus, and also there are descending projections
down here to brain stem.
So we're beginning to get quite a good idea
about which brain areas are connected to which other brain areas,
and that's obviously of key importance
to get a causal and mechanistic understanding
about how the mouse brain processes sensory information
and how that might lead to behavior.
So in order to link what we've learned about the mouse brain with behavior,
we clearly need to study mouse behavior in quite some detail.
One of the more interesting things that the brain can do
is to learn and remember things,
and an important behavioral paradigm, then,
tests learning and memory,
and one of them is the Morris Water Maze.
In the Morris Water Maze experiments,
a mouse is put inside a swimming pool
that's been filled with opaque water.
The mouse will then swim around and eventually it finds
that there's actually a hidden platform
where it can stand on, and swimming is a rather exhausting task for a mouse,
so it's rather happy when it gets here to the submerged platform
where it can then stand and relax,
and gradually, over days, as the mouse is put in
it learns to swim more and more directly to this hidden platform
guided by visual cues that are placed around it.
So in this example, it'll learn to swim more and more directly
towards the star, and that's how it'll locate the hidden platform.
On a transfer test day, the platform's removed
and the mouse is again placed inside the swimming pool
and it swims around, and if it's learned correctly
that the platform's located here, in this environment around the star,
it'll swim around in this area here
searching for where that platform might be.
So one can then quantify the amount of time
that the animal spends in this target platform
relative to the other quadrants, so you see okay,
it might spend a lot of time here in the target quadrant
and much less time in the three other quadrants,
and then we can quantify to what degree the mouse has learned the behavior
and remembered it.
So that's a way in which we can test learning and memory in mice.
Now, we can connect that
to the specificity of the genetics that we've been talking about
and in 1996 Susumo Tonogawa and his laboratory
created a rather interesting mouse
that tested some of the things that we've been discussing.
When we were discussing glutamate receptors,
we had time to think about long term synaptic plasticity
and at that time we said that probably was the basis for learning and memory.
One of the key experiments that supported that
has been this experiment here, where genetic knockouts,
deletions of NMDA receptors have being carried out
in highly specific brain areas.
So we discussed that the NMDA receptor during the concomitant activity
of presynaptic release of glutamate and postsynaptic depolarization,
the magnesium popped off the NMDA receptor
allowing calcium to enter, and that would then start off plasticity processes
that might then encode memories.
In this particular experiment, the Tonagawa lab created mice
where the LoxP sites were surrounding the NMDA receptor type I gene.
So this is the one that's absolutely critical for NMDA receptor function.
They put LoxP sites surrounding the NR1 gene,
in the presence of Cre-recombinase that would then knock out the NMDA receptor
but in other cells that were not expressing Cre-recombinase,
this would be wild type because the LoxP site
is in introns, and doesn't affect the expression
of the native NMDA receptor.
The Cre-recombinase was expressed in a highly specific way inside the brain.
It was expressed post-natally and only in a small part of the brain
called the hippocampus,
so there's a so-called CA1 region of the hippocampus
that's located around here, and cells in this area are known to encode space.
So they are so-called play cells
that were discovered by the 2014 Nobel Prize winner,
John O'Keefe, that encoded the location of where the animal is in space,
and it's thought that the reason that the animal learns
these place representations is through long-term synaptic plasticity.
They can then use that spatial information to navigate
and find where it is in terms of carrying out this spatial memory task.
So when the Tonagawa lab made these CA1 specific
NMDA receptor knockout animals,
they put them through the Morris Water Maze learning
and on the transfer test day, the animal would swim around
and have no recollection that the platform would be near its supposed location
here, in this example, by the star.
So learning and memory was disrupted by knocking out NMDA receptors
in this highly specific area of the mouse brain
thought to encode the spatial location
and that then gives us strong genetic evidence
for the role of synaptic plasticity in NMDA receptor function
in forming memories.
So that's one interesting behavior
that's been studied in some detail in the mouse,
and begins to give us confidence that we are on the right tracks
in terms of understanding simple aspects of mouse learning and behavior.
Making neurophysiological measurements while mice are swimming
in the Morris Water Maze is relatively difficult,
so investigators have also been working on simplifying the behavioral tasks
and also providing a more highly-controlled environment
in terms of the sensory inputs that the mouse will receive.
One of the key steps has been to develop head restraint mouse behavior
where metal implants are placed on the head of the mouse
and that then stabilizes the mouse brain to the level that there are only
microsecond level movements of individual nerve cells.
So we can make high quality neurophysiological measurements,
electrophysiology and imaging of the mouse brain
and the mice rapidly adapt to this head restraint behavior
and they show no obvious signs of discomfort.
So in one particular paradigm we've investigated sensory perception
through these whiskers.
So we put small pieces of metal on the whisker.
The animal is placed on top of an electromagnetic coil,
and we can give one second pulses onto this whisker,
and the animal learns that those one millisecond deflections of the whisker
predict reward availability if the animal licks a reward spout.
So here we can begin to study mechanisms of motivation,
reward based learning, and sensory perception
in a highly controlled manner.
And we can get to that causal and mechanistic understanding
of simple behaviors in a head restrained mouse.
Other laboratories are making
more sophisticated head restraint mouse behaviors
where an animal might be running on a floating trackball
and navigating a virtual visual environment.
So there's a great deal of excitement in the neuroscience community right now
about the head restrained mouse as a way of linking genes' behavior
and neurophysiology at that level where we might get
a causal mechanistic insight as to how the brain really works.
So there's a great deal of interest in understanding the mouse brain
from a purely scientific point of view
where we're really addressing one of the main frontiers of human knowledge.
Another major reason for studying the mouse brain, of course,
is to try and understand the mechanisms of brain dysfunction
with the hopes that we might be able to provide cures
or at least new treatments,
so brain diseases make a big impact on the world,
both at the level of individuals and for society as a whole.
So a key goal for neuroscientists is to develop better treatments
for brain diseases,
and clearly, in order to improve our ability to repair the brain,
it's really helpful to understand what's going on inside the brain.
So a challenge for many neuroscientists
is to see if we can develop rational therapies for brain diseases.
So in the field of so-called translational neuroscience research
we might have a genetic mutation that is linked to a human brain disease
and you put that genetic mutation inside the mouse.
There we can study the causal impact of that genetic lesion
and see what difference it makes to the brain cells,
their connectivity, and also their function during behavior.
One can then begin to develop therapies that in the first instance
work for that mouse model of the disease,
and if that works in the mouse, one might then think of seeing
whether that also works as a therapy in monkey models of the same disease,
and if we have these two scales over which that treatment works,
then, of course, it becomes extremely interesting
to try and see if we can translate that to humans
and the hope, of course, is then that we provide rational therapies
for the large number of brain diseases that cause such trouble for mankind.
And so in this video we've seen that understanding the mouse brain
might provide clues as to how the human brain works.
Investigating the mouse brain has been helped by large scale efforts
in terms of molecular biology, gene expression patterns,
also in terms of connectivity and general organization of the mouse brain.
And so, research in the mouse is developing at a rapid rate
and there's now growing excitement about the ability to connect
that basic fundamental knowledge about proteins, molecules, cells,
wiring diagrams, with the behavior of the mouse.
We're now beginning to see the causal and mechanistic underpinnings
of behavior at the level of cells and synapses
by studying the mouse.
Over the next videos we'll look at more of the details about how, technically,
we make measurements from the mouse brain during behavior
and how we can also manipulate and control
the neurons in the mouse during behavior.