HOW TO MAKE A BRAIN

LECTURE 9: INVERTEBRATE NEUROGENESIS

There are 5 pre neural genes in invertebrates, these genes are hited by hlh transcription factors and then generate
the whole neural system.

PNS (Periferal neural system):

➢ Atonal genes (ato and amos): Yamins work: Much earlier it had already identified the preneural genes that
generate mechano sensory brisels→ later the ATOnal gene was found. Some larvae were mutant when it
came to ATO gene and when they took a single hemisegment of the anterior region, they lacked all the
chordotonal organs, the stretch receptor organs were completely missing.
Also found that atonal mutants have coordination problems , the adults also lack all the photoreceptors of
the eye.
CONCLUSION: The atonal is the preneural gene which generates the chordotonal organs and photoreceptors
of the eye.
If you look at the brain region corresponding to the eye development, it generates without any problems.
To show the expression of the atonal gene, they ectopically induced the expression of atonal nerve by
adding it to the gene, since on the wing of the fly you have a few chordatonal in the hinge zone, when they
over expressed on through this region they generate.

ATONAL is a master factor to convert cells into neurons. Atonal in the wing induces chordatonal organs but not the
photoreceptors, since the wing does not express the hox genes which generate the photoreceptors.

➢ Achate and scute

The acute scute homologue and atonal homologous genes encode for basic helix-loop-helix transcription factors,
which bind to a common partner (DAUGHTERLESS) one will cleave to daughterless and nind to DNA, regulating
transcription of genes that lead to neural fate. These factors typically bind to 6 base sequences on the DNA,
depending on what factor it is depends what middle bases it binds.

Neural ectoderm (the cells compete one another, winner cell converts into the neuroblast) → new nervous system

The neuroblast comes out the neural ectoderm, they delaminate and move inside the cells that lose out, the rest of
cells will form the cuticule. The same mechanism acts in the outer mesoderm. Thanks to this mechanism of cell
signaling one another there are some cells which will form a sensory organ and will tell the rest not to form a
sensory organ but an epidermis→ Lateral inhibition.

➢ Pronural genes→ give proneural cells competency to become neural, Basic-helix-loop-helix transcription
factors.Mutants Not produce neuroblast or sensory organs.
➢ Neurogenic genes→ mediate cell-cell interactions to prevent neural competency in precursors. Encode for
the Notch signalling. Mutants produce too many neuroblasts or sensory organ precursors. EX: mutants
become very hairy (excess in sensory organs).

There is a group of cells up-regulating a proneural gene, all of the cells hae the competency to become neuroblast
or sensory organ, one cell starts having higher levels of the proneural gene→ inhibit the proneural gene expression
in neighbouring cells.

Lateral inhibition involves Delta-Notch Signalling:

Proneural gene→ expression of a ligand known as Delta. The receptor of delta is Notch.
When there are the same levels of delta this signal to Notch in another cell→ both signaling equivalently one
another. When a cell have higher levels of Delta, it starts its own synthesis of Delta expression and then has a
stronger signaling of Delta-Notch receptor. These cells end up self-regulating the proneural gene and the first cells
end up knocking off the expression of the preneural gene.

The cell which end up being the neuroblast migrates inside and the surrounding cells become the epidermis. The
type of neuroblast it becomes depends on where it was within the neuroectodermal, due to the pattering genes
which provide the neuroblast its particular personality. The nerublast divides itself and generate ganglion Mother
cells, which end up generating neurons (Hox genes).

Each neuroblast has its particular identiting depending on where they are ubicated within the chordal cordon. All of
the neuroblast use the same Temple clock to determine which ganglion mother cell they are gonna be generating.

At first high levels of Hunchback transcription factores→ They decrease→ 2nd mother cell is going to inherit the
lower levels→ the cell is going to inherit Kruppel→ then PDM-1 → Castor.

Extricnsic cues from the epithelium determine the side at which ganglion mother cells bud off from Neuroblasts,
this occurs exactly parallel to the underlyingepithelium, the plaen of division ensures that the ganglion mother cell
buds off absolutely opposite to the underlying epithelum. The spindle fibres which divide chromosomes are sligned
perpendicular to the underlying epithelium.

Certain proteins are only inherited by the neuroblast of mother cells. There are two types of proteins.

➢ Proteins aligned with the spindle complex.

➢ Numb→ ends up getting pushed away from the epithelium region and ends up in the dorsal region of the
neuroblast. Numb ends up in the Ganglion mother cell. The GMC is now only able to divide once more to
generate Neurons. Numb makes the cells which have received it to switch off the notch receptor.

When the two cells resulting after division end up having diferent proteins, this means the two cells will follow
different life pathways. After each cell division the cell will make new numb and deposit it onto the gmc. The
neuroblast will go into qiescent phase after generating GMC.

When the GMC divide, two equal cells are generated, one will receive numb but the other one wont receive numb,
in the cell that cell that recieves numb, that will bind to the notch receptor making it unresponsive to the delta
signal from its sibling cell. In most cases, the cell that recieves the numb is the one becoming the neuron, the other
one actually goes through apoptosis.
Ryu’s work: Numb mutants were not completely knocked out, they were jus thypomorf. In the original numb
mutant, when they looked at the second division, both of these cells became 2A cells because of the lack of numb,
during the second division, these mutants ended up with 2 hair cells and 2 socket cells.

When you overexpres numb, both cells

become numb+→both of the two cells
inherit cells, there for they are both 2
B cells and then both of them end up
producing neuron and seath, 2 of each,
these individuals look bald.

After this, a strong numb mutant was

discovered, this individual didn’t
produce numb at all so after first cell
division non of each cell can produce
numb, both of them were numb- and
notch ON. After the second division
both of the resulting cells are numb
lacking so them become soacking cells.

➢ Numb because of the mutants→ numbt to the touch.

LECTURE 10: NEUROGENESIS IN VERTEBRATES

The neural stem cells spam from the ventricle to the peel surface, each cell has contact with the inner and ventral
side of the neural tube. The lumen is where the cells divide.

Individual stem cells can generate all of the cell types within the nervous system→ EX: Scientist labelled a single
stem cell with a fluorescent mark, then, that stem cell produced more cells and then, the motor neurons,
oligodendrocytes and astrocytes generated by that single stem cell were labelled with the fluorescent.

Interkinetic nuclear migration: Cells migrate

from the lumen to the surface during the G1
phase, they duplicate their DNA during S phase,
the nuclei migrates back down to the luminar
phase. Then during the mytosis the cell duplicates
and form 2 nuclei, the cell goes through
cytokinesis and then there are two new stem
cells. In lots of the stem cells they also retract the
cell cytoplasm so they’ll divide, but others don’t
do that and keep their citoplasm conections in
both luminal and peel surface.

This process occurs quite quickly.

If we take a look at this process happening in a mouse, we will see that on the ventricular zone start expressing
genes related to the glia cells,they divide here and at some point they generate basel brogenitors, cells prodcued
by asimetric division and then migrate away and divide in generate mature neurons which will be sat on the mantle
zone.

During the early stages cells start dividing symetrically. Then they atrat dividing asymetrically because of the way
they divide in the plane of division relative to the surface, there are proteins set down in the stem cells more in
one region than in the other. Starting from the stem cell, then the expressed proteins define in which cells they are
becoming.
If the stem cell divides perpendicular to the ventricular lumen both of the daughter cells receive all of these
proteins so this is a symetrical cell division which create two identical stem cell. Later on the plane of division
occurs paralel to the centricular surface, which makes cell division asymetric, then each cell recieves different
concentration of these asymetric proteins, one of these cells becomes a basal progenitor, and the other one
becomes an apical neural stem cell. The basal progenitor has NOTCH receptor and the apical stem cell has
NUMB/NUMB LIKE protein.

The proneural genes are activated in the Stem Cells an expressed due to de activation of the previous expressed
genes (Sox2, Zic and Hox). There are several transcription factors which are important fror regulating the
neurogenesis:

➢ Proneural:
1. ASH1
2. ATH1
3. NGN
➢ Diferentiation: (making neurons)
1. NeuroD
2. NeuroM
➢ Pro- oligo: Provide identity
1. OLIG2→ helping to make oligodendrocytes.
➢ Inhibitory: astrocytes
1. HES: stop the expression of proneural genes.
➢ Inhibitory HLH: antagonize bHLH. Prevent the neural genes from turning the neural crest cells
too quickly.
If we take a look at the spinal cord, we can see that Atoh1 is expressed mainly in the dorsal region, Ngn1 is mainly
in the ventral region and Ascl1 is expressed in the dorsal region in between. So each region of the neural tube has a
different type of gene expressing in there in order to develop a different type of neurons.

INVERTEBRATES vs VERTEBRATES:

In Drossophila for example, the proneural genes get expressed in the ectoderm on neural ectoderm and the
proneural genes will then generate neurons. There is lateral inhibition mechanism, and will turn these other cells
into non neural precursos and they will make ectoderm and epidermis.

In mammals the proneural genes do not switch on until all the cells that are going to be signal between are already
neural stem cells, so the proneural genes ONLY ACT over neural stem cells and start making neurons. The
delta/notch signals also occurs in vertebrates such as in invertebrates, but a strong signal of these factors is
neeeded. If not, these cells will not revert to ectoderm THEY WILL STAY AS NEURAL STEM CELLS, that differs from
invertebrates.

If you over express the proneural gene you get neurons developed really early and turn many of the stem cells to
make neurons. In the vertebrates system there is a good equilibrium making having a good ammount of proneural
genes activated but not too amny stem cells turned into neurons.

If you get a loss of function,a big ammount of glia cells and astrocytes start appearing. That is telling us that the
proneural genes stop the appearance of too many glia cells within the nervous system.

Same mechanism than in vertebrates, mutual inhibition activates the HES system as in flies. All of the stem cells
produce an ammount of the proneural gene but then one of them will go and up-regulate it so starts producing a
strong delta signal to its neighbor, then activate the harry nhacer split (HES), and when it is up regulated it inhibits
the proneural gene, but that cell will kep being a neural stem cell, not as in invertebrates.
Same mechanism that in invertebrates.

One of the main problems generated by this mechanism is that all those cells upregulating HES due to the light
delta-notch signal would become glial progenitor cells, this is a problem cause then there wont be any cells able to
generate neurons later. There are processes upregulating these system so the body is able to make later cells of
the nervous system both neurons and glia.

There is a mutual inhibition, the HES gene in vertebrates, inhibits its own oroduction by linking to its own
promoter. Cells are constantly in a state of flux capable of generatinf Basal progenitors or Gliogenic progenitors,
the levels of hess rise and it comes a point when they fall.

➢ HES produces its own production and the porneural gene→ cross talk with one another.

In irder to make neurons stem cells has this oscillation going, it gets to a point when the nucleus comes back down
to the lumen and it is about to divide, it gets just a kick of enough signal and produces higher levels of the
proneural gene and then the system needed to transform cells into nerons is activated.

Due to this delta-notch signaling all the cells in the epithelium are in these constant state of flux where the levels
of the preneural gene gets oscilalting.

Once the stem cell has enoguh amount of preneural gene it will activate a cascade of genes which allow the cell
entering the proliferation phase and then go into differential phase and produce neurons.

1st→ enough amount of preneural gene.

2nd→ proliferation phase.
3rd→ blocking of the proliferation.
Basal progenitor switch from proliferation to differentiation:

Sox 2 expression preceeds the expression of sox 21, it binds to every gene that sox 2 does and inhibits them, it
helps cells to differentiate.

➢ In the neural crest, when the WNT gene levels are sightly higher than the BMP genes the cells start
producing neurogenic 2 will go onto forming sensory neurons for the peripheral nervous system, if the BMP
signal is sightly higher then they form ganglionic cells of the autonomic nervous system.
➢ Looking at the placode regions, preneural genes and neurogenic genes are important for determining the
correct number of neurons. Looking at the cochlea, for example, there are 3 rows of hair cells and also
support cells, lookin at a mutant mouse when it comes to the Atoh1 gene, it only has hair cells.
Proneural gene important for generating neurons, similarly delta-notch lateral inhibition is important as
well, then the proneursl gene is expressed in too many cells. Similarly if you block HES1 and HES5 there are
too many neuron.
1- Proneural mutant→ loss of neurons.
2- Delta-notch signal loss // blocking of HES1/HES5→ too many neurons.

LECTURE 11
Group migration:

The cells use one another to migrate from the subventricular zone into the olfactory bulb.

Migration which leads to the cerebral cortex:

Pallium→ cortex

Sub-pallium→ striatum

The stem in the pallium generate cholinergic neurons and the ones in the subpallium gabaergic neurons. If we look
at the development of the structure in the ventricular zone cells divide simetrically at first, then they divide
asimetrically, post-mitotic neurons migrate radially to form a preplate at outer surface, then at the cortical plate
stage newly generated neurons migrate along radial glia and form the cortical layer between yhe preplate
neurons.CP layers contain pyramidal cells.

Inhibitory neurons come from the outer layer of the sub-pallium, cells migrate tangentially into the cortex and
interdigitate with the pyramidal cells.--> GABAergic inhibitory neurons.

Cerebellum development:

➢ More complicated

The dorsal region of the neural tube is going to form the roof plate and the ventral region the basal plate. Cells of
the roof plate push the adjacent parts of the neural tube laterally. Looking at a cross section a structure named the
rhombic lip can be seen.

The cerebellum is derived from two separate proliferative zones, Purkinje cells generated in the ventricular zone
migrate radially from the cerebellar plate, then the Granule cells arise from the Rhombic lip the external granule
cell layer divides and then the granual cells migrate in the opposite direction of purkinje cells and sit in a region
underneath purkinje cells, they assume a T shape, these cells contact many purkinje cells but only one dendrite of
each purkinje cell. **

The cerebellum works out the movement control

Cerebellum have several inner layers.

An important protein is involved in this laminar formation, this is called Reelin (RELN) initially discovered in mice.
When mice can synthesize reline protein they showed defects in cortical organization that results in poor
coordination and cognitive impairment. Reelin is expressed from the **** cells. When you don’t have reelin the
order of the layers change.
Migration in hindbrain:

Somatomotor neurons do not migrate and project their dendrites, they are generates in pMN. Nonetheless,
brainchiomotor and VISCEROMOTOR neurons don’t stay where they are generated within the neural tube, they
migrate to more lateral regions from the floorplate.

Neural crest migration:

Migrate all the way out, as the neural tube develops the mesoderms develops as well. The neural crest cells
migrate due to the chemorepulsion by the posterior somites.

LECTURE 12: GLIOGENESIS

During development some stem cells stay as stem cells. However, there are some other stem cells which express
enough of the proneural gene to reach the threshold that is now going to allow them to start becoming a basal
projenitor to make neurons, when you reach this the proneural gene drive various signals and then cell regulates,
then it depends on the signals which the proneural gene drives which path is going to be followed by the cell.

There are some stem cells which never reach the point of differentiation since the peak of the ASCL1 oscillation
doesn’t coincide with the cell division so never reaches the threshold necessary to switch to basal progenitor (BP),
the expression of HES genes are favoured and then regulate the glial-ness of these cells in order for them to
become glial cells, they are still stem cells because they express high levels of Sox2 and are in a proliferative state.

These cells can, at a point, reach the proneural threshold and start making basal progenitors to make neurons or
starting to upregulate HES.

Looking at the image it can be seen the fact that stem cells talk to one another through lateral inhibition or mutual
inhibition, neurogenic signals will just be secreting morphogens that are helping to upregulate proneural genes
within the neural stem cells to let them make neural progenitors. At later stages, development switch over and
then stem cells are gonna be start making the glia,the neurogenic signals weaken and gliogenic signals start
becoming predominal.

From Neuron to Glia Production Jak-STAK:

The main gliogenic signals within the brain are part of the JAK-STAT signaling pathway, when it comes to early
stages, the neural stem cells are going to be generating basal progenitors to make neurons meanwhile through later
stages they are going to start making glia. It was shown in experiments that mice which lacked from the JAK-STAT
signalling components (LIFRbeta, gp130 and STAT3) developed impaired astrocyte differenciations, showing the
importance of JAK-STAT signalling pathway in order to switch between neuronal to glial cells.
Proneural genes actually lead to de silencing off the genome for genes as STAT-3, for example, allowing cells to
make neurons. In later stages, when Jak-STAT genes predominate, the neural stem cells develop astrocyte. The
factors that developed neurons secrete help to upregulate the jak-STAT genes within the neural stem cells.

The neural stem cells are going to produce glia cells when there are enough neurons.

One of the main factors producing this whole transformation is cardiotrophin-1, binding to the same receptors at
leukemia inhibitor factor, the neuros release it in vivo to trigger de JAK-STAT pathways for neurons to change from
neurons to glia. This factor binds to LIFR-beta and STAT-R co protein, then activates it and causes the
phosphorylation of the Janus Kinase, once it happens it leads to the phosphorylation of STAT3 and then it dimerise
and enter the nucleus, binding to the genes and upregulating GP1300, Jak1 and STAT3, and also HES which is going
to help the cells switching to becoming glial progenitors.

Fate determination in Neural Crest:

From somite 7 down to somite 22 in there are going to be neural crest cells generating somatosensory neurons of
the peripheral nervous system and also ganglionic neurons of the autonomic nervous system. Firstly neural crest
cells generate sensory neurons and then go on to make the Schwann cell glia of the peripheral nervous system,
support cells that will be wrapping myelin around the somatosensory neuron axons.

Neuregulin 1→ leads to glia. In the neural crest the neurons will be generating neuregulin 1 and tell tha arriving
neural crest cells to form Shwann cells. That shows neurons go before glia.

Migrating Neural cres cells→ Dorsal Root Ganglion → differentiate as Neurons (Nrg-1) → later neural crest cells
encounter high levels of Ngr1→ Ngr1 interacts with JAK-STAT and GLIA cells are formed.

Retinal stem cells:

There is a zone in the di-encephalon which is called the zone limtans intrathalamica which expresses sonic
hedgehog, this inhibits Pax 6 and separates the Pax 6 domain in left and right eyes. These neural stem cells migrate
out in what is going to be the retinary.

At the time in which neural stem cells start dividing asimetrically to form neurons, it is showed that with each
division different types of cells are developed being.

1. Retinal ganglion cell.

2. Horizontal cell.
3. Amacrine cell.
4. Cone photoreceptor.
5. Bipolar cell.
6. Rod photoreceptor.
7. Mullar cell (GLIA)

The very last division is when it generates the glia cell. Art each division the stem cell will change some of the
transcription factors that it is expressing so by the time it divides to generate the next cell this cell will inherit
different transcription factors, allowing to become the next cell one different from the previously generated.

During development there are a lot of switching on oscillations of different transcription factors.

LECTURE 13: NEUROGLIA

The glia as concept was born in the middle of XIV century, glia would mean glue in ancient Greek, something which
provides oily influences, in fact, the glia is the oily connective tissue of the brain, in which neurons are embebed.

Golgi→ Found cells in the brain with star like appearance, Sending signals and processes to the blood vessels
through special structures called ‘’end-feet’’ , providing an interface between brain parenchyma and circulatory
system, recollecting intuitions from the blood and distributing them through the brain.

Theories of the CNS organization:

Neurons are separated entities and integrate information. Carl Ludwig Schleich came with an important idea about
the glia cells, he was the founder of the theory of neuronal-glial interactions. He suggested that the glia-cells
because of their propensity to change their volume, they interfere with synaptic transmission processes, being
capable of causing a coma or a full general anesthesia.

Ramon y Cajal came up to similar ideas, he deduced astrocytes were important for the regulation of many
physiological processes, astrocytes, for example, could change the diameter of the blood vessels and that way
regulate the flow. Astrocytes can also interact with synaptic transmission and he identified this process as a
mechanism for sleeping.

Neuroglia: heterogenous population of non-neurons cells providing a supportive role, being responsible for brain
homeostasis, myelination and defenses.

There are two different types of neuroglia, Central nervous system neuroglia (macroglia and microglia) and
peripheral nervous system neuroglia (shwann cells, olfactory ensheathing cells, enteric glia and satellite glial cells).

There is almost the same number of neurons as glial cells within our body, what varies is the distribution of these
cells.

Evolution of neuroglia:

Its connected to the evolution of the nervous system, belongs to animalia kingdom, they are not represented within
the diffuse nervous systems, there is no evidence of supportive cells. When the fist ganglia emerged, first proto-
astrocytes were born, if you kill those cells in an animal, it survives but with difficulties.

➢ C.elegans: bilateral organization , with a neural ring, several glia cells are the very first example of proto-
astrocytes controlling the regulation of this neural ring.
➢ Leech: Multiple ganglia fused forming the fore-brain each ganglia contains 400 neurons and also contains
glia cells regulating , specific cells covering the somato of many neurons. Pair of giant glia cells in the
middle of the ganglia responsible for the regulation of the synaptic connections.
➢ Insects: In Drossophila there are found different types of glia. The ensheathing glia cells which are forming
the barrier, in insects the brain hemoring barrier is made by astrocytes. There are also cortex glial cells
which cover tons of neurons. Finally there are astrocytes like cells sending very different types of
organization processes being responsible for all the homeostatic control of synapsis of Drossophila.

Within invertebrates there is a quite big diversification of glia, coming to cordata a new type of glia was born,
which is called the Radial glia, appearing with the layered organization of the brain, they form sacaffolds through
which the pro-neurons move and attain their relevant positions.

Within mammals glia cells acquire the full responsibility of the homeostasis of the nervous system.

The highes ratio of glia/neuron cells can be found in big mammals. However it is true that in the process of
evolution a complexity of glial cells have increased in humans, in humans astrocytes are ten times larger and
complicated, they support about 2 million of synapsis. Within human bodie there are also specialized astrocyte
(laminar, polarized, etc.)

Astrocytes:

Thee name comes from late XIX centuryintroduced by Lehnosshek, many glial cells stained due to the gold
technique , gold stains cytoskeletons and membrane, that’s why he gave them the particular term of astrocytes.
Actually astrocytes have a more bush form.

Non neuron cells which contribute to the homeostasis of nervous system and provide defense of the central nervous
system, all levels of organization, from molecules (pH, water, etc.) synapsis and forming neurons network, they are
involved in the glymphatic system, they can percieve the levels of oxygen, CO2, etc. They regulate the responses
needed. Basically all levels of homeostasis.

There is no specefic marker for these type of cells. There is also a huge heterogeneity of astrocytes when it comes
to morphology and functions. They can have a moshy morphology like the protoplasmic astrocytes, or maybe have a
neural appearance as the ones making the spinal liquid.

They have many different functions:

1. Developmental.
Embryonic neuro and gliogenesis. Glia cells come from the stem cells divided in an asymetrical form, some
of them don’t move and stay in a sub-ventral zone, forming the radial astrocytes, fully responsible of adult
neurogenesis.
Astrocyte interact within synaptic contact and release many different substances controling
synaptogenesiss, for example colesterol.
2. Structural:
Define the basic arquitecture of the brain since each protoplasmic astrocyte in grey matter occupais an
independent territory and all neuronal elements within that territory are covered by the astrocyte→
Neural-glial-vascular unit.
3. Metabolic:
Potassium buffering, potassium is a basic element of synaptic transmition. They put the potassium back to
neurons, they take it up by the sodium/potassium boms. They regulate the water movements with
aquaporines.
4. Control of CNS microenvironment: Important for the vascularisation of the brain, form the feet and covers
every part of every vessel→ blood brain barrier, in the perifery there are gaps between cells covering
vessels. In brain vessels those cells are connected, there is no gap.
Important in the system which cleans up the brain, linfatic system main function is provide water in order
to clean other system. In the brain there is no lymphatic system, but there is other system regulated by
astrocytes and its fucntion is to provide water to clean up the brain of bacterias, virus, etc. They have a
vascular space filled with water, through this the water can be flown through the parenkyma of the brain.
This is important for sleeping because the cleaning of the brain occurs during this ssystem working.
5. Signaling

The brain is of course incredibly vascularized organ, one of the most vascularized. Whenever neurons are active the
blood flow increases as well, this is known as functional hyperaemia, the brain possesses an intrinsic mechanism
by which itrs vascular supply can be varied locally in correspondence with local variations of functional activity.
The more blood comes the more water is moved to the hot areas.

Astrocytes create neurovascular units and link synaptic activity to the tonus of brain capillaries thus regulating
microcirculation.

Most of the energy demanded by the brain comes from restoring the levels of ionic substances, mostly sodium and
potassium. Glucose is equally distributed among astrocytes and neurons. Nonetheless some of the astrocytes are
capable of breaking the glucose and provide with more energy to neurons. Most of the energy demand comes from
synapsis.

Glutamate→ Astrocytes→ glycolysis→ breaking of glucose→ lactic→ imported to neurons and produce energy.

Homeostasis regulated by astrocytes:

Astrocytes are also endocrinal cells so they produce about 200 molecules of all sort kinds. Supported by 3
mechanism

➢ Exocitosis
➢ Diffusion
➢ Transmembrane transport

They are essential for sodium homeostasis, they produce a sodium sensing machinery built around atypical sodium
channel (8X), activated when the level of sodium goes up.

The sense CO2 within blood→ exaltation of calcium

Muller glial cells act as light guides in the retine, they detect hiw kight is coming, absorption for a particular
waylines.

One of the most important functions of astrocytes is the synaptic regulation, indispensable, 60% of all synapsis can
not be carried out without glia cells. The bush-like form of astrocytes cover the synapsis. In addiction, the
separation and isolation of each synapsis is carried out by astrocytes as well. Astrocyte monitories how synapsis
behace, theyneliminate the dysfunctional synapsis.

They sense what neurons do because of their huge number of neuro-receptors and channels. After these receptors
are activated they TRIGGER the polarization and electrical impulse, they are electrical non excitable cells,
however they are chemically excitable and can affect in electrical signaling. In fact, calcium kis the molecule
governing the astroglial function. Nonetheless, sodium is a molecule for fast neuronal-astroglian signaling, there
are substantial sodium fluxes, very important since the majority of astrocyte transport is regulated by sodium
dependant transporters (pH, neuroglial signaling, secretion of molecules, glutamate).

Neurotransmitter homeostasis as glutamate, released by neurons and taken up by astrocytes by the sodium
dependant transporter, the astrocyte transform glutamate into glutamine and its passed to membrane. That is very
important since neurons can not synthetize glutamate de novo. It is the same with GABA which has to be synthetize
from glutamine as well.

Neuroglia and neurological diseases:

Neuroglia are also defensive elements. About 600 phisological forms of diseases, that is because the brain is a very
complex system. Maybe by interacting with glia cells, which regulate the homeostasis of nervous system new cures
to neurological diseases can be found. If the disease is characterized by a homeostatic failure then the main reason
for the disease is a fail of neuroglia cells. Neuroglia failure is the universal pathogenetic step in neurological
diseases.

➢ Neurons become stressed, they have a bv very low capability of defending.

➢ Neuroglia cells express important changes (function, morphology, etc.) when they are attacked, Gliosis→
specific reaction of astrocyte for pathology. There is a whole class of pathological changes happened in
astrocytes.
There are also situations in which astrocytes become the drivers of the disease, they lost their whole functions and
they lose their ability to protect the homeostasis. In many diseases of the CNS astrocytes acquire reactive
phenotypes, hallmarks of which are increase in GFAP expression and hypertrophy processes. Maladaptive
astrogliosis can be a therapeutic target.

COVID 19: Ways to affect the brain. Neurological symptoms (dizziness, sleepiness, etc.) pneumonia→ ability of
bring O2 to the brain→ metabolic processes→ wrong excitation of cells. Systemmic inflammation in which the
blood start having a lot of inflammatory factors.

LECTURE 14:

Starting with terminology:

➢ Oligodendrocyte: cell with few processes, myelinating cell of the CNS discovered by Pio Del Rio Hortega.
➢ Schwann cell: myelinating cell of the PNS named after Theodor Schwann. Analogue of the oligodendrocytes
in the PNS.
➢ Myelin: means marrow in Greek, myelin is a complex of membranes joined by proteins. Secreted by
Schwann cells and covering the axons.

The myelin sheath is many concentric layers of cell membranes around the axons, each of these structures is called
lamella, the thickness of these are between 50 and 200 layers, they are connected with myelin proteins, about 70%
of lypids and 30% of proteins, these myelin proteins appear only in vertebrates. Invertebrates have similar
structuresbut not myelin.

The classic functions of myelin is to increase the axon potential propagation, this is not an electrical signal. The
axon potential is propagating opening and closures of ion channels, in fact, two channels, sodium channel and
potassium channel. For the axon potential propagation to occur it is needed the change of the membrane potential,
this happens thanks to the entering of positive ions into the cell, this process create an electromagnetic field which
is sensed by a neighboring channel, then it happens a movement of single electrons then it activates the gate
proteins and the gating mechanism can happens,

In a non-myelinated axon what happens is there are equally distributed through membrane and this way the
potential propagation occurs through the channels.

In a myelinated axon the myelin covering divides it into different segments, there are internodal segments, in
which the myelin sits, and then there are Nodes of Ranvier, which are the segments of the axon in which there is
no myelin covering. What internodal segments do is prevent ions to go through the membrane under the myelin
sheath, each node of Ranvier has sodium and potassium channel in a very high density, what happens is an
electromagnetic field is created in these nodes and that field can be felt by the next node, which would respond to
this electromagnetic field and make the potential propagation to happen faster since the signal jumps from Node of
Ranvier to other Node of Ranvier.
 Jumping saltatory propagation

Myelination decreases the diameter of axons needed for the same AP propagation velocity, in principle it is an
alternative strategy, for example, invertebrates have thicker axons and more channels since they don’t contain
myelin, they have a similar structure but without myelin, they just have thicker and more layers.

The association of joints forming a connection within the human brain is called the human connectome. The human
brain is unique since in a very small volume it has nearly 200 billion of neurons cells and 100 billion of non neurons
and 100 trillion connections forming the connectome. The brain can be interpreted as the largest supercomputer in
the world.

Organization of the PNS:

There are sensory nerves, which go to dorsal root, motor nerves, originating from ventral roots and the sensory
nervoussystem also involves sensory neurons sitting in ganglia, most of them in dorsal root ganglia. There are other
ganglia in the head like trigemical ganglia.

There is autonomic nervous system, which is divided in sympathetic and parasympathetic, they all originate since
the brain, and they both have ganglia, so parasympathetic and sympathetic nervous system have their two neurons
and autonomic ganglia from which axons go to the periphery.

There is a big nervous system which populates our gut, this is meters long and have their own autonomic enteric
nervous system, which is quite big, containing more neurons than the spinal chord. It is consired to be a second
brain since it controls the gut.

The peripheral nervous system originates from the neural crest, part of the neural tube which detaches very early
in development. Then it is divided in several parts, from these parts all the PNS is originated.

All the neural crest cells are pluripotentand turn into committed precursosrs and then precursors for neurons and
glia, the precursors for glia can originate Schwann cells, inmature Schwann cells, non-myalinating cells, etc. from
migratory glial precursors olfactory epithelium is biorned as well.

Types of Schwann cells:

There are three types:

➢ Myelinating Schwann cell: major type these type of Schwann cells can be found along the axon. A
single Schwann cell forms a single internodal region→ 1 to 1.
➢ Non-myelinating: Covering up to 6 axons, they do not secret myelin.
➢ Peri-synaptic Schwann cell: Covering the synapsis between neurons and muscular fibers. Very small
body with a huge membranal sheath coming around the axon and forming the internodal region. They
expres special substances like caveoli or rafts, instrumental to make the myeline sheath. Originated
together with muscular junction, they migrate with the axon, they are obligatory for the formation of
 neuromuscular junction, the shcwann cell completely covers the synapsis. There are a lot of
pathologies related with the destruction of this synapsis.

Schwann cell and axon are in constant communication, the axon releases a lot of signals and ATP which si send
to schwann cells, the ATP triggers the Schwann cell and makes it to send metabolic substances, Schwann cell
are supporting the axon.

Ganglia:

Very specific glia cell sitting in the ganglia, thin cells which are covering neurons, connected with GAP junctions,
forming a functional sincitius by which they interchange molecules, substances, electrical potential, etc. Every
neuron in the ganglia is covered by a satellite glia cell (take care of glutamate, deffense→ neuropatic pain,
homeostatic balance, etc.).

Myelination in the central nervous system: Oligodendrocytes:

Cells with few processes, up to 4 processes, wandering around and seeking active axons to cover it to star its
myelination, up to 40 axons, the larger is the the axon, the higher is the number of lamella.

They develop from neurepithelial cells, they turn into radial glia, it asymmetrical divides and then form
oligodendroglia precursing cell or OPC. These unmature oligodendrocytes have to migrate, in a newnborn brain the
vessels are not covered yet by oligodendrocytes, it takes time for the myelination process to happen.

They migrate along blood vessels, the astrocytic coverage hasn’t happened yet, astrocyte have to come to the
vessels and produce a coverage, then the migrating OPC meets the vessel and sense a signal which tells the
oligodendrocyte that part of the brain is already under a barrier and that cell starts covering the axon forming a
mature oligodendrocytes.

The oligodendrocytes have to acknowledge which axons are nearby and active. The mechanism works by the firing
of axon potential secreting potassium out of the cell, this potassium is quite high, only the dendro-glial precursor
cells have this special channel which sense the very small potassium signal then the oligodendrocyte understands
this is an active axon and goes to cover it and produce myelin. This process takes years.

Myelination is related with plasticity.

The oligodendrocytes have also the function of eliminating the excess of potassium secreted during the axon
potential.

➢ Esclerosis→ Demyelination diseases in which oligodendrocytes are the main target of T-cells from immune
system. Oligodendrocytes being destroyed.
Most of these oligodendroglial precursors are really peculiar such as the NG2 glia was discovered in the 1980s, NG2
stands for proteo-glycan which is expressed in these cells and it is used as its marker. Central nervous system, very
complex and very elaborated processes, which involve moving, similar to microglial. They are not really glia,
properties similar to neurons, but also contact blood vessels, neuronal cell bodies and RECEIVE PROPPER SYNAPSIS
FROM NEURONS. These are the only glial cells which receives proper synapsis.

Some of them can differentiate into oligodendrocytes, they are highly proliferative, only in an advanced age the
proliferation stops.

Enteric nervous system:

Includes the guts, mainly the intestine, in which there are many muscular layers, and then, inside them the mucosa
and the plexus. Mienteric plexus and mucosa plexus, accumulation of ganglia. Between those two plexus sit the
glial cells, they make a very intriqued mesh, they are similar to astrocytes (take care of glutamate, potassium
buffering, etc.) they have many receptors.

There is a very special type of cells, the mucosa glial cells, then there are the intramuscular, between muscles, the
intraganglionic and the fibrous between ganglia. All these glial cells contacts the mucosa. Inside the gut there is a
special environment.

Glia cells contact the mucosa→ inside the gut there are many microbia, a lot of bacteria in a symbiotic
relationships→ if these bacteria are killed this affects the body→ glia cells form a BARRIER which takes care of
different microbial life trying to enter the organism and damage us.

Olfactory nervous system sits inside the brain. Axons down to glomerular layers and olfactory bulb, axons are
covered with olfactory glia which cover very intimately, with a lot of interesting properties→ PLURIPOTENT STEM
CELLS. When they lay on the olfactory bulb nothing special happens but when you take them away they become
pluripotent stem cells which can be injected to the brain they may generate neurons.

Microglia in physiology and pathophysiology:

Special cells which take care of the defenses of the central nervous system. They are cells with different
morphology among them. They have non-neuron origin, they originate outside the brain in the very early stages of
development, the end of week 3 of gestation, they go into the blood when there are non blood barriers, they
change completely their shapes within the blood vessels, small body and display many elaborated processes.

Around birth they still multiply, they make an invasion of the whole brain and disseminate through the brain and
spinal cord shortly after birth, they migrate within the blood vessels. They are constantly moving in such a way that
a single microglial cell scans their environment once every hour. They identify synapsis, they have an incredible
number of receptors→ immune receptors, acquire neural receptors. The receptors change with age, sex, and also
their presence change depending on the part of the nervous system they are in.

They make a networking with other cells in the brain.

Functions:

➢ Not looking for pathology but important for a proper development and function of the brain→ invade very
early, they are no other glia cells, they support early synaptogenesis.
➢ They shape synaptic connections. If something is wrong with synapsis microglia cells detect it. If they don’t
do it properly there would be an excess of synapsis. When synapsis is not working they detect it. → some
types of autisms the main reason is the microglial cells not shaping the synapsis properly.

In pathology, if a very local pathology occurs the microglial cells perceive it and go to the area of damage and
plaster it in a very small amount of minutes. Complete process of reaction, they produce a big deal of reactive
phenotypes in order to protect the brain.

Microglia can be divided in subtypes, N1 and N2→ wrong.Not bad and good, there are just several types of
phenotypes.

LECTURE 15: SIGNALING

There are channels known as ionotropic receptors:

➢ Purinoceptors
➢ Glutamate

The nervous system is aproperty of animalia kingdom, starting from comb jellies til Bilateralia. In ctenofora and
cnidaria there is a difuse nervous system, neurons distributed through axons, in bilateralia the situation is
different, neurons do not situate homogenously through the body, they have a concentration in the brain.

Neurons descend from epithelial cells, these epithelial cell endow with all important machinery, iron channels,
receptors, transporters. The beginning of very first neurons was possibly specialized peithelium cells.

Evolution of central system→ centralization.

1. Nerve cords.
2. Nerve ring→ fused ganglia
3. Segmentally divided ganglia.
4. Star fish→ 5 raium with one nerve ring connected

Neuroglia becomes obligatory for nervous system survival in bilateral animals. Radial glia→ new type of glia in new
type of brain in deutherostomes.

C. elegans→ one of the most studied animals on Earth. 56 glial cells 50 cover dendrites of sensory neuron, 4 of
them envelop the nerve ring. Sensory glia of C elegans seeks around the body and form specialized organs→
sensillium, which allows them to sense the environment.
In platyhelminthes there are two nerve cords connected to a ‘’brain’’→ accumulation of ganglia, on the brain there
are multiple types od neurons, there is also a specific neuropil glia, also a peripheral glia assisting the brain.

Tartigrada!--> survival strategy: DEATH. Extremely small, characterized by an incredible perceptibility and
adapatation. They have ganglia and peripheral neurons.

Annelida→ Segmental animal, with segmented ganglia.

There are a lot of diversity of glia among the animal kingdom. Drossophila: divided in peripheral nervous system
and central nervous system. Inside the rbain there are several types of glia (Ensheating, cortex, perineural,
subperineural, etc.).

In mollusks the neurons sit inside the glia. In Octopus there are half a billion of neurons covered with many type of
glia, probably up to eighty type of glia molecularly and 70 morphologically.

Verebrates:

Different glia cell types, responsible for all the homeostatic processes inside the brain.

Evolution of blood-brain barrier:

Mechanical fence between the brain and the rest of the body, in insects for example there are different type of glia
which make this barrier, this barrier is ONLY glial.

Crustaceans→ fully glial.

Vertebrates:

➢ Teleostei: The blood-vein barrier is made by epithelial cells, many layers of perivascular glial cells.
➢ Chondrostei: the epithelium cells are not connected and the blood brain barrier is endothelial, parenchyma
is part of this particular structure.

It started as an extension of glial barrier.

Myelin:

The white matter is myelin which is incredibly important for the functioning of the brain.

LECTURE 16: AXONAL GROWTH AND GUIDANCE:

Axonal growth is a field that appeared at the on the 70’2, 80’s , 90’s. There are complex events involved in axonal
growth (cytoskeleton reorganization, organelles, molecules secretion, etc.).

These depends on early neurogenesis when axons are programmed to do all of their functions. Many aspects of
axonal growth have much to do with the migration of cells, when they project their axons when they move to the
external layers of the brain when developing.

The growth cone is essential fort he process of axonal growth, they are the driver seat of this process.

➔ Experiment in
which they
change the
orientation of
the frog’s eye
 ➢ EX: They cut the optic nerve between the retina and the optic chaism of a frog and they observed the
axons kept navigating to tectum from the growth cone.

Key experiments from the 70’s in which they took the eye of the frog, in which the retinal is divided in different
areas with different ganglia cells that projects their axons towards the tectum, the key experiment that was done
with this mapping was invert the map, they observed the projection of the axons changed their direction as well
and behaved as the original positions, theu re-establish the original connections.

Chemmoaffinity hypothesis:

The mechanical answer is because of the chemoaffinity hypothesis, the axons only grow toward an essential target
tissue, and neurons have the ability to detect chemical molecules from the target tissue and project their axons
toward them. In the temporal area of the retina, for example, there is a FA3 receptor, but these receptors is not in
the nasal area, the protein of the signal can bind to this receptor is Ephrine A2, which is generated in the posterior
part of the tectum but not in the anterior, Ephrine A2 repels FA3, so this neurons would not be able to grow here.

Growth cone: inside it there are microtubules and actine,they are both organized in different ways. It is rich in
receptor molecules, they receive signals of the environment.

The growth machinery works depending on the different signals cached from the receptors. The different
components of the cytoskeleton work in distinct ways, meanwhile actine filaments are going backward the
microtubules are constantly extending into the periphery og the cell and collapsing back. If the cell receive a signal
the microtubules can stabilize and move toward a direction, then, the actin part is moved to the new tip.

If we take away the F-actine the microtubules cannot divide and then the axon grows straight and faster, but the
axons wouldn’t be able to respond to signals.

➢ Actine→ directionality
➢ Microtubules→ growth speed

If you take an axon and put it on fibronectin it hits a barrier of proteoglycan secreted by glia cell, this means a
inhibitory signal and the axon would not grow through that substance because it is a signal for neural injury. If F-
actine is toxified the axon would not be able of grow away but they still would not pass through the CSPG, they just
stole.

The directional stabilization of microtubules induces growth cone turning. If you use different substances that
affect the microtubules polymerization this event may be observed.

➢ Nocodazole: inhibits the microtubule polymerization, so the growth cone is biased against the direction in
which the nocodazole is placed.
➢ Taxol: stabilizes microtubules polymerization, so the direction taken by the growth cone is changed by this
substance. ( Stabilization of microtubules→ turning of the growth cone).
➢
➢ Taxol + cytochalasin D: taxon stabilizes the microtubules but the cytochalasin D is an inhibitor of F-actine
so there is basically no spaces for microtubules to go to the right and the effect of taxol is inhibited.

Part of these early expression of genes during development kos related with
axonal growth. Within tissues there is an expression of certain guidance queues
laid out in spatial temporal patterns, then, the system can start interacting. When
they start drawing out they have different receptors on the surface which will
attract or repel these different signals. Through a code of expression of specific
sets of receptor molecules in the neuron it will respond differently to the
environment in which it navigates.

➢ EX-1: Motor neurons sit in the ventral horn and arrange in different motor.
There are some neurons which go from one side of the neuron system,
they go ipsilaterally within the nervous system.
➢ EX-2: The commissural neurons are across the floorplate meanwhile the
plate motorneurons are staying ipsilateral, this behaviour is maintained by
the different chemoaffinity of these neurons. et-1 is expressed at the
floor plate and moto neurons express rRobo-2, Robo2-positive neurons are
repelled by rSlit-1 secreted in the floorplate but the same rSlit-1 does not
repels commissural neurons.
This was proved because they took explants and put them in culture, they
observed slit repelled motoraxonal growth of ventral horn, meanwhile
when they took an explant of the commissural area this did not happen.
 ➢ EX-3: Sema3A is expressed in the middle of the limb bud area, it repeals the growth of cones, separating
them into a ventral and dorsal path. If you have not sema3A motoneurons start growing everywhere.

Signaling into the cytoskeleton:

The backflow of the cytoskeleton consisted basically in actin filaments polymerizing from their plus end and
depolarizing from the minus end, whereas microtubules are growing from the plus tip and then also depolymerize
from the plus tip. So there is a dynamic instability, in a cell there are quite a lot of microtubule binding proteins
which can bind to the plus tip of microtubules, along the lattice of microtubules and work as motors running along
the molecules, it is the same with actine, so a regulation can be made by the action of a lot o binding proteins
which regulate this dynamics.

The key insight is actually in F-actine. There are other key factors as Rho-GTPases which are shared signaling
effectors and orchestrators of actin dynamics, they are inactive when they are bound to GDP, this GDP can be
changed by GTP, then activate the protein and allow it to bind to the effectors, called GAPs, which hydrolyze GTP
into GDP again and inactivate the Rho-GTPase.

All the signaling pathways for axonal growth work because of the action of GTPases, so basically there is a some
common systemic code by which different factors communicate with the same proteins.

This different GTPases are binding proteins can change the dynamics of the cytoskeleton.

Within the growth cone biomechanical processes are happening, the different forces can affect directly into the
cell behaviour.

➢ Aplysia: It has huge growth cones, about 50 micron in diameter. apCAM is a molecule which can stick to
cells and is also expressed by growth cone, apCAM binds apCAM and therefore activate the beads ( surfaces
in which the actine backflow is happening). F-actine is moving toward apCAM in the surface in this beads.
Further experiments showed that at this site apCAM accumulates where the beat is, at the same time
srkinase is accumulating in exactly the same site as a dependance of holding the beat back, driving the
microtubules to this direction. If the activity of src-kinase is deactivated, then the microtubules will not
keep growing toward this place, they are not responding, the mechanical action of the actine binding to
apCAM, apCAM binding th the kinase and the beat being hold back recruits a sr-kinase.

Application of axonal growth into neural injuries:

When you get an injury one of the main problems may be inflammation, which is extremely damaging, if you apply
taxol that reduces inflammation and axons can grow better across this site.

Another type of Inhibitory cues are coming from glial cells, they form cspgs and inhibit axonal growth,
olygodendrocytes do not get properly removed, which differentiates them from Schwann cells in the periphery
which are more dynamic. Myelin components are also inhibitory for axonal growth.

➢ EX: Exposed growing axols to MAG and, which is a myelin associated glycoprotein and puff it only from one
side, the axons had a tendency to grow on another direction. If you puff from MAG, suddenly, integrines get
distributed on the left side and non on the right side. MAG binds to the Nogo-R receptor, and this receptors
talks to Rho-A which means it tracts F-actine, but, at the same time it is working a pathway by which
through Pi3Kinase and calcium signaling they triggered clathrin mediated internalization of integrines and
also a collapse of the system.

LECTURE 17: MAP FORMATION AND TARGET SECTION

➢ Information in the CNS is grouped modality specific domains.
➢ The terminal projections of axons within a specific modality, are arranged in a topographic map of the
peripheral location of the sensory neuron cell bodies.

Modality specific projection.

Within fly larvae, there are brain, opctic lobes and neural chord, and then 2 different segments with a group of
neuronal cell bodies, axons of sensory neurons project to the CNS based on location of the neuronal cell body in the
periphery, so the ventral dash sensory neurons projects their axons together in the segmental nerve→ example of
modality. You have bipolar dendrites, sensory neurons,e tc. All the sensory from lateral and dorsal cluster project
together into the intersegmental nerve (ISN).
 ➢ The axons project to the CNS based on peripheral locations of the neurons cell body and NOT due to
the neuron sensory modality.

When these enrves enter into the CNSthe different modalities of neurons project into an specific region called
neuropile, in one region you have a modality.

There are three main regions the external sensillum (tactile), the chordatonal (golgi tendon organ) and the multi-
dendritic (muscle spindle) occupying three different tracts.

The external sensory neurons tract actually cross the midline (midline in general).

Modality specific projection:

In the fly, the modality specific projection can happen due to the proneural gene that sensory neurons expression
of different axon guidance receptors. One factor very related to all this process of signaling is Robo3.

The external sensory neurons are produced thanks to the neural gene ASC, they have ASC identity and because of
that they do not produce Robo 3. They project into CNS in create a signal of slit secreted from the midling
preventing them to produce Robo 3. The terminal projections ARE NOT repelled from the midline, then cross the
midline.

It is the opposite with proprioceptive neurons(ch) their projections are repelled from crossing the midline as they
enter the CNS.

Within the CNS the terminal projections of neurons find their particular domain due to their MODALITY rather than
where they are located initially.

During the metamorphosis of the flies most of the larval sensory neurons degenerate, but one neurons always
remains and acts as a pathway that the adult specific neurons follow. For ex, for segmental nerve, the new sensory
neurons send their axons into the CNS and follow the segmental nerve, which was the ventral nerve in the larval
system, the domain for legs 1 and 2 are in a ventral position up in the brain.

Wings are dorsal so they follow the intersegmental nerve which were prefigured from lateral and dorsal neurons in
larval system. Come between leg1 and 2 axons and occupy a domain dorsal to leg domains and then go out into a
posterior tract.

Looking at the frontal right leg of a fly there are chemosensory hairs, 4 neurons in the base detecting signals thse
send axons into the leg neuromere or neuropile occupying a central domain called PROTOTHORACIC NEUROMERE
T1. There are similar for the other legs. In between chemosensory there are mechanosensory bristles which respond
to touch, all the mechanosensory neurons send their axons into the CNS and occupy a domain where the
information is going to be processed. Third type of sensory neurons, the proprioceptive monitoring stretch which
has the same mechanism of modality.

Different types of information end up in the same part of the CNS to be processed one another.

Somatotopy:

Axons of a particular modality terminate in the neuropile in a topographic map that relates to the location of the
neuronal cell body in the periphery, that is called spatial mapping.

Looking at tactile domain, there are tactile neurons in the posterior and anterior bristles, they project in to the
posterior and posterior part of the neuromere respectively. Within neuromere there is a map that matches the
location of the cell body as it is in the periphery. Spatial mapping of the periphery in the CNS.

MAP FORMATION AND TARGET SELECTION:

➢ Modality-specific domains and topographic maps are present throughout the CNS of vertebrates
➢ somatotopic mapping is present at all processing levels in the transit of information from periphery to
cortex
➢ Topographic mapping is based in gradients.
➢ Ephrin-Eph gradients map retinal ganglion cells in the superior collicus.
Modality specific projection:

Vertebrate sensory spinal afferents in the dorsal root ganglion show modality-specific mapping in the dorsal horn of
the CNS. In the periphery (limb) there are different type of receptors (nociceptors, mechanosensory, etc.) The cell
bodies of all these neurons are in the Dorsal root ganglion and send axons out to innervate these structures, and
then a projection in to de somatosensory cortex. These different modalities send their collateral branches into
specific domains within spinal chord.

Proprioceptive neurons, two of the factors needed for this specific

modality projection are Runx-1 (Transcription factor) and Runx-3.
Runx-1 runs the expression of one receptor different from Runx-3.
Nocioceptic neurons are Runx-1 expressing neurons, if ectopically
applied Runx-3 instead of projecting to CNS then deviate and then
target a deeper layer of the dorsal horn. They are guidance factors
which control the modality of axons and neurons.

Topographic projections:

Looking at all type of sensory information a topographic map can be

seen, in the frontal lobe on the very back end there is the
precentral gyrus→ motor coordination information, upper
motoneurons controlling particular muscle occupying a place in the
cerebral motor-cortex next to a nearer muscle in the body.

Certain parts of the body have more discrimination for touch, for example, there are much bigger representation
on the cortex for the hand than for the back.

➢ Everything somatosensory is mapped in the brain cortex. Nociotopic, Tonotopic and Visuotopic.

It is the same in other animals, happens the same with mouse whiskers, the sensory neurons innerving whiskers
project in to the CNS creating a somatotopic map.

1. Whisker to brain via trigeminal nerve.

2. Brain to thalamus.
3. Thalamus to cortex.

Looking at the cortex as a whole, there is a FGF8 source of morphogen being secreted from the dorsal anterior
midline and BMP signals from the dorsal-posterior midline. (same signaling when pattering the cortex). These
gradients lead to the expression of transcription factors Pax 6- anterior, Emx1 and Emx2- posterior. There are three
region observed in the cortex→ pattering.

Changing the levels of those morphogens within the cortex causes the shift of the map, rather than the terminal
projection from one particular modality and occupy its domain now can spread or get compressed. If Emx2 levels
increase then the visual cortex expands and the somatosensory gets shifted rostrally. Altering FGF8 changes the
map as well, when you reduce it the map shifts rostrally, when increased the map shifts chordally, and when
express from the posterior (chordally) a duplication of the primary somatosensory cortex.

The projection of the axons are working out where they should go thanks to these gradients.

Retinal system:

Topographical projection of retinal cells in the tectum, the temporal retinal ganglion cells go to the anteriar region
of the tectum and the nasal to the posterior. Looking it dorso-ventrally, the dorsal retinal ganglial cell go to the
lateral part of the tectum and ventral to the medial part. This is worked out by different factors called Ephrins.

Gradient of Ephrin B highest medially and lowest laterally. The Retinal ganglia cells express receptors for EphA and
EphB, the Ephrin S-EphA is a repulsive gradient. Any retinal ganglion cell expressing high levels of EphA will NOT go
to a part were high levels of Ephrin A are presented. There are complementary gradients of ligand and receptors
being Ephrin the ligand and Eph the receptor.
 ➢ Retinal neurons expressing high levels of EphA receptor, express high levels of Ephrin A ligand, that I swhat
happens with the nasal receptor. The signaling is not one directional. Nasal cells have high levels of ephrin
A and they actually avoid high levels of Eph Aand end up going to the posterior, in this case they are using
the reverse ligand.
➢ The retinal neurons in the temporal region express high levels of Eph A so they repel Ephrin A in the
posterior part and end up in the anterior. They are doing this through a forward signal, usual signal in
which the cell with the receptor is responding to the representation of the ligand.
➢ Cells expressing intermediate levels end up occupying an intermediate region.

There is also an attraction gradient. In dorsal region there are High levels of Ephrin B get attracted to high levels of
Eph B, this is a reverse signal. In the ventral region the high levels of EphB expressed in this retail ganglion cells
will be attracted to the high levels of Ephrin B in the medial part of the tectum.

OLFATORY BULB

Not SOMATOSENSORY.

Projections of olfactory neurons in the antennal lobe represent an olfactory map rather than somatosensory mapIn
simple worms such as C elegans single neuron express multiple receptors meawhile in mammals single neurons
express a single receptor, and each projects to 2 out of 1800 glomeruli.

In absence of neurothrophin you cannot activate CREB.

Srcond factores prmoving survival in neurons are CYTOKINES. (LIF, CNTF, IL). These factors bind to the receptor
and form a complex which then activates the Jak-STAT kinases and help neuron to prevent apoptosis.

In neural stem cells stop cells from making GLIA and not NEURONS.

Hormones: main- testosterone→ more generated in males→ converted by aromatase in estrogen. tHis has a
receptor and Estogen receptor enter the nucleus and regulate the genes.

Hypothalamus can also trigger apoptosis in other regions with this same system. → contect dependant action.

Showed in hamsters→ applied in a critical stage and testosterone is presented in 1h time …

APOPTOTIC PATHWA:

➢ BAD protein→ key factor.

➢ If there is no factor binding extracellularly→ no phosphorylation of the receptor→ Non phosphorylated BAD
protein.
 ➢ BAD stops BCLX from binding to other factors.
➢ Citocrom C →APF-1→

LECTURE 18: EARLY PROGRAMMING

Any tipe of environmental experiences if keep acting long enough, they perturbe the brain homeostasis and affect
to neurodevelopmental reprograming, thanks to epigenetical mechanisms. → REPROGRAMING.

The long term effects depend on:

➢ Reprogramming mechanisms
➢ Type and severity of exposure
➢ Timing of exposure
➢ Sex
➢ Effects on germ cells.

Different windows of susceptibility during brain development due to changes in grey matter, hormone levels, etc.

Epigenetics is the mechanism underlying the process of changes happening in the brain due to the environment, a
gene can be silent or expressed depending on the signals received from the environment, there are no changes
inside the DNA but in how it is read. There are different ways of modifying DNAs lecture.

➢ Metilation
➢ Histone modification
➢ Synthesis of non encoding RNAs

These three type of modification cannot happen at the same time.

DNA metilation:

Addition of the methyl group in cytosine by the DNA methyltransferases (DNMTs), then cytosine becomes 5’ Methyl-
cytosine. CpG islands are areas of the DNA where we can find several of this cytosines and are really prompt to get
metilated. The metilation induces tramnscriptional silencing.

Histone modification:

There are 5 different type of histone modification that can take place. They are reversible, allowing for the
dynamic control of DNA expression, also there are post-transcriptional. The most common is histone acetylation, is
basically adding acetile to the histone by histone acetyltransferases, when this groups are added then the form of
the chromatine so it relaxes and it is easier to be read.

There is another type of modification, histone methylation, which is mediated by methyltransferase.

Histone phosphorylation is not that common, it plays a rol with DNA damage response but it is not quite clear in
mammals.

Non-coding RNAs.

There is a lot of RNA compared to the amount of RNA which is transformed to proteins. This RNA is not really
useless, there are long ncRNAs (>200 kb) and short ncRNAs. This ncRNAs can interfere with transcription inducing
chromatin modifications or even affect splicing, editing of RNA, affecting to mRNA stability, etc.

These epigenetic modification change the chromatin, changing it from repressive to permissive.

PARENTAL STRESS:
The brain is plastic and its development can be changed by external stimuli. Stressor is any factor that changes or
threatens to change the homeostatic balance of an organism. The stress can be of many types and affect to
different areas of the brain, creating different responses.

There are two different areas of the body that can get activated depednding on the kind of stress the animal is
exposed to, there are 2 axis whether the response is fast or slow.

Hipothalamus-pituitary-adrenal axis: The first stage is the paraventricular nucleus of hypothalamus, releasing
glucocorticoids, such as CRH or AVP, these go to the pituitary gland releasing ACTH to the adrenal cortex, and this
stage is really important because from here, glucocorticoids are secreted in order to shut down this response to the
hippotalamus, there is a NEGATIVE feedback.

Cortisol: is a hormone that specifically stimulates hepatic gluconeogenesis, inhibits glucose uptaje, protein
degradation and facilitates lypolisis. It has anti-inflammatory process and immunosuppressive auto-inflammatory. It
is released when exposed to good levels of stress. When having levels of cortisol too high it can lead to chronic
stress which is dangerous for the body.

The parental factors also have an influence into the developing of the brain, the stress of the mother goes to the
baby (levels of glucose, cortisol, insuline, etc.) If the placenta experiences changes in levels of different substances
such as cytokines, nutrients transports and receptos, etc. that can affect the baby. Maternal stressing during
pregnancy has long-term effects on the offsprings future stress sensivity , reward and cognition. Effects depend on
gestation timing of the exposure.

➢ Decrease GR in hippocampus.
➢ Increased CRH inamygdala.
➢ Increased corticosterone production after acute stress.
➢ Cognitive deficits inspatial learning and memory tasks.

There are different responses to h¡why people tolerate differently the stress. If you experience higher levels of
stress in childlife and during adulthood there are good chances to get mad, the same with no having stress at all.
When looking to animals, a difference is observed, when an animal that did not experienced high levels of stress
during early life then experiences it in adulthood, start showing clear patterns of disease.

The three dimensional model has three axis, the levels of stress in early life, the levels of stress experienced in
adulthood and the programming sensitivity, which depends on different factors.

LECTURE 19: EPIGENETICS

Introduction (porque no entiendo que hace esto aquí:

Obesity is defined as having a body mass index or BM1 above 30, in the last century the environment has changed so
much and now the biggest problem is obesity, around 700m of obese people, the percentage of obese people has
increased above a thirty four percent since 1974. Obesity is linked to lots of different diseases, it reduces life
expectancy depending on the sex of the person.

Making a brave resume, the body weight

depends on how much you take and how
much you waste. Normally we have an
homeostatic system secreting hormones,
like leptin, insulin, ghrelin, etc.
controlling the hunger and making sure
we eat only what we have to waste.
However, there is another player, the
hedonic system, refers to a reward
system, linked to a part of the brain
activated when we enjoy food, this
system is specially triggered by high fatty
food, high sugary food, etc. This system
can be affected by early life
experiences.
Nutritional/hormonal environment acting hand to hand with genetic predispositions. There is evidence that if a
baby is exposed to bad food, either early life or post-natal life this can affect structural and functionally the key
metabolic organs and then, the metabolism of the individual, being more prompt to suffer from diabetes,
metabolic diseases, obesity, etc.

These long-term changes are mediated by epigenetic mechanisms, this has been proved by different experiments.

➢ Maternal and paternal overnutrition: you give a rat or a mouse high fat diet for a long period of time, then
the rat gets pregnant and we can observe epigenetic changes in the offspring, reward pathways in the brain
get altered, there are also changes in the adipose tissue since it triggers pro-active adipogenic genes.
This can be triggered by paternal overnutrition as well, the offspring, in particular the female offspring
show changes in genes involved with pancreatic-beta-cell functiton.
It is not only the maternal obesity, paternal obesity as well.

Experiments about other fields as famine have been carried out. In the beginning of the 20th century there were
several famine periods. Boys before puberty suffered from famine during a great period of time, then children and
grandchildren of these boys had greater risk of suffering from cardiovascular diseases.

Maternal transmission:

The main explanation for this event is the maternal transmission of information. If a pregnant rat is exposed long
periods of stress, different kind of machinery start acting, such as cytokines, machinery affecting oxygen levels,
etc. This will hve a major impact on the fetus.

Due to this change of environment experienced by the mother, the offspring id going to have a long term change in
behaviour. If the fetus is female, she will have germ-cells which are going to be exposed to the stress levels at the
same time as F1 generation an the cause major changes in F2 generation.

This is not transgenerational pathway since them both have been exposed to exactly the same environmental
changes. To be a truly transgenerational, the next generation have to inherit that particular phenotype from the
parents even though they have not being exposed to the stressful environment. For a male this is the 2 nd generation
and for a female, the 3d generation.

Paternal transmission:

In a lab, male rats and mice are rarely related to the offspring. If there is any paternal influence it has to be with
the sperm. There is some evidence that actually that can happen, there is some of paternal transmission. When you
somite male rats to high levels of stress (drugs, bad diet, dominant rats) this can have a major impact on the
programming. If the male rat impregnates a female rat then the offspring expresses a certain phenotype coming
from paternal site.

Spermatogenesis takes around to 6-8 weeks, around this period the sperm can experience changes, one it is
finished then the chromatin compacts and during this period there is no chance of any changes. Then the mature
sperm is in the epididymis a new window to induce changes opens and induce epigenetic modifications. The idea is
that there can be also paternal transmission of epigenetics but the window of opportunity is much smaller than it is
in females.
Postnatal stress:

One of the most studied. The studies mainly focus on the

experimental paradigm, ‘’when the female rats give birth, they
have these maternal instinct that will provide lot of licking and
grooming to the pup and feed them’’. Early experiments have
found that naturally, just spontaneously some female rats that
provided licking and grooming to the pub while others didn’t care
about the offspring, this showed that the level of grooming.
There is a protein that has shown to play a critical role in terms
of post natal stress, rats that do not provide a lot of grooming to
the offspring, low levels of this protein (NGFI-A) in hippocampuns
means the promoter of the gene will be methylated, thant ends
up in the silence of that gene. The GR gene is silenced and then
means low levels of low levels of glucocorticoid receptors in
hippocampus→ the pups show higher levels of corticosteroid than
normal and higher levels of anxiety behaviour.

The situation with rats providing high levels of licking and

grooming to their pups is totally different.

➢ Ex: If pups are born in day one they only spend 20 days with their mothers, during those 3 weeks if they
removed the mother from the nest several times for a long period this caused a lot of stress to the pups
(don’t have fur, they cant see, cant eat, etc.). The experiment showed these pups showed higher levels of
stress hormones in hypothalamus, lower levels of CRH.
➢ Ex: fragmented maternal care: When there are insufficient nesting materia this cause stress and the
consequence is the decrease of CRH in the hypothalamus, if these animals experience stress→ decrease of
CRH expression in hypothalamus.
➢ Prenatal stress: Increased CRH expression in hypothalamus.

Conclusion: Stress leads to alterations in the HPA access and the type of alteration seems to depend on the timing
of the stress.

LIGHT

Light is an important regulator of hormone secretion due to circadian rhythms. A studies showed that preterm
babies exposed to constant light when incubating them, had disturbance biological rhythm, they need longer times
to gain weight and the visual development was affected by the constant light.

The season when you are born ca affect sleep patterns, personality and mental health as well.

The circadian system regulates hormones, body rhythms, behaviour, etc. based on the light levels and works with
the day For example, body temperature is lower in the morning and it increase during the day. The light is
detected by specialized receptors and goes to a particular area in the brain called suprachiasmatic nucleus, the
SCN is the principal biological clock, every single cell have circadian rhythms regulated by the SCN.

➢ Melatonin: sleep hormones helps us to sleep better.

➢ Afternoon: greater cardiovascular strength, best codination, etc.

Most of the research of the effects of light has been made in rodents. Mice pregnancy period is around 21 days, the
next 3 weeks the pups are lactating they are with the mum. The pregnancy period correspond with the first
trimestry of pregnancy in humans, 10 first days of a mice would correspond to the second trimester of pregnancy,
the third trimester is the next 10 days and 8 weeks is when a mouse is considered a young adult.

In rats and mice, the photoreceptors of retina develop from birth and are mature to day 15, at level of the SCN
there are several things happening, such as neuron formation, which happens during pregnancy, synaptogenesis and
formation of glial cells progress lowly from pregnancy until 2 week after birth, there are a lot of things happening
and ending their comformation during the first 2 weeks of life, that’s why this is a critical time for mice to be
exposed to stressful environments.

For the first weeks of life everything is rather inmature in the pups, the mother is the one detecting changes in the
environment and passing the information to the pups. The influences of the mother becomes weaker by the time
pups can receive information due to the circadian system is fully developed.

➢ EX: external light exposure has a major effect in the offspring during the first 2 weeks of life in some areas
of the brain , it can also affect peripheral clock of different organs and can affect the whole system. These
animals suffer alterations in how they respond to stress. Clock is a particular circadian gene, if mice were
kept in constant darkness and then moves to normal cycles they had normal sleeping pattern. However, if
they were exposed to constant light, despite putting them back to normal light cycles they showed same
phenotype as humans suffering of delayed faced sleep syndrome.
If you raise normal mice they show normal sleeping pattern, they do not show the mutation of clock gene,
this is telling us the genetic background of the individual can play a major role interacting with the
environment.
➢ Other experiments took a look at in which season the animals were born, spotting differences between
winter and summer. Mice raised during summer were more prompt to show anxiety behaviour.
➢ In humans they found that there were more than a hundred studies linking schizophrenia and season of
birth, people born in winter were less prompt to experience schizophrenia later in life, and also people
born between hanuary and may were more likely to develop bipolar disorder. However, schizophrenia is the
one which has a stronger link with seasonality.
➢ There was another study with adult humans in which they found different levels of grey matter density in
specific areas depending on when they were born, summer and winter. Male showed differences in superior
temporal gyrus and females showed differences in several parts of the brain.

Conclusion:
LECTURE 20: SYNAPSE FORMATION
Synapyogenesis is a dynamic situation where neurons put out new rights with an axon, or become dendrites, finding
their way to a target to become stable and non-mobile in order to form a functional synapsis.

DYNAMIC NATURE OF THE MOBILE GROWTH CONE→ FIXED RIGID STRUCTURE OF SYNAPSE

Two main techniques for studying the development of synapsis:

➢ Morphological and anatomical analysis in vivo to see how the structure of synapse appears to be changing
over time.
➢ In situ imaging of the processes that take place to form a synapses and how it changes over the time.

This studies give us an idea o the speed of these processes. In terms

of the general development of synapsis this is one of the last stages
of the CNS development. If we take a look at the subcortical visual
pathway in the retino-collicular pathway, we could see that until
RGC axons reach the SC and then they start to form sinuses and
then the first synapses start to occur in the SC at the end of that
period of the developmental pathway.

We can observe there is a relationship between the density of neural

body cells and the dendrites and glia appeared in the nervous
system, there is an initial contact and then when everything is ready
you can have a massive proliferation of connectionsin order to try
and achieve a function.

➢ Firstly we can observe a rapid increase in neuronal cell body

density after birth, this may cause the increase in the
amount of synapses per neuron.
➢ Then the neuronal cell body density decrease with time and
the amount of dendrites and glia start to increase.

Taking a look at the associated changes in synapse structure along the mouse anterior ventral nucleus. There is a
view of how axon connections may look like over the time, initially a single synaptic connection and then these
proliferate into a lot different connections→ changes in the actual ultrastructure of the synapses.
When looking at early stages of life it is difficult to
define the synapse, in fact there are presynaptic
vesicles but there is little to do with post synaptic
density, but when getting into adult stages, it can be
told if it is an asymmetric synapse, excitatory
synapse, etc. → changes in terms of features and
elements that support synaptic transmission.
Maturation process.

EXPERIMENTS: SPEED OF FORMING A FUNCTIONAL CONNECTION

Using a spinal low motor neuron forming a functional connection with the muscle in order to execute its function.
Ebrionic spinal motor neurons, using a patch electrode, make high resistance seal onto one of the malleable
membrane , pull off a part of the membrane since it is the postsynaptic target spiny neurons and is going to have
receptors on it that can potentially be activated by any synapsis.

This acts as a system in which it can be detected wether the neurotransmitter that would act on those receptorsis
being released or not. When the patch is introduced next to the spinal neuron growth cone it start to show inward
currents suggesting that the AChR (Acetyl choline receptors) have been activated-→ release of Ach→
demonstration of the system being finally tuned.

You can also demonstrate this with a similar situation with central nervous system neurons. You can demonstrate
the growth cones are ready to release neurotransmitters culturing these neurons on a special substance called FM-
64, a membrane binding fluoropore, then neurons are provided with an extra amount of potassium which would
make the membrane potential increase and then FM4-64 is taken up by endocytosis following vesicles fusion into
growth cone and filapodia, but then, the potential would be reestablished and the neurotransmitters reabsorbed by
the neuron again, this may be a sign of the neuron being a functional vehicle, this is demonstrated because of the
appearance and disappearance of fluorescent caused by the medium which the neuron has be exposed to.

The fluorescence imaging shows there are discrete vesicles that contain the fluorofore→ complete cycle

There is another way of using this culture system, using immune

aside chemistry to prove the presence of postsynaptic receptors.
There are NMDA-receptors, AMPA-receptors, Presynaptic vesicle
markers. There are evidence of both pre and post synaptic
vesicles present and colocalization, which is telling us synapses
are happening.

INITIAL CONTACT

One of the main signals of the contact process is that there’s not only pre-synaptic signaling but post-synaptic
signaling as well. Taking a look at the neuro-muscular junction, you can load the spinal moto neuron with calcium
fluorophore→ spike of presynaptic calcium in presynaptic neurone, and as a consequence changes happen.
Main sight of fluorescence coincides with the presence of calcium in the presynaptic neurone.

Calcium signaling helps to organize and stabilize the cytoskeleton, which makes the growth cone to remodel:

➢ Actin polymerization to stabilize proto-terminal

➢ Cytoskeletal reorganization to access secretory vesicles.

Evidence of regulation by retrograde signalling the secretion of Netrin-1 acting on its receptor the DCC,

There are also changes occurring on the post-synaptic target, manipulation of the spinal neuron with an electrode
and observe if there is activity or not, observe what the consequence of contact is for that postsynaptic neuron.
Firstly there is a spontaneous activity after the stimulation but after some minutes more activity is registered what
suggest more neurotransmitter is released. It is the same with the muscle fiber, the activity changes over time in
terms of their amplitude, changes occur.

Adhesion after contact in neuromuscular junctions:

In terms of neuromuscular junction, adhesion rapidly follows contact,

this fact could be demonstrated by a mode, there are not only
functional changes but strength, and FISICAL change in the structure.
Using a muscle fiber cell as postsynaptic target and a neuron we can
measure the strength of the adhesion by displacing the myoball away
from the site of contact, observing how the neuron sticks to the
myoball after 15 minutes the strength of adhesion can be measured,
you can even get complete displacement of the growth cone.

The time frame is very rapid, in terms of 10 minutes interaction it gets

adhered to the growth cone.

Intercellular signaling is very important in synapse formation, when you culture pontine neurones they send axons
pathfinding and engage one another going searching for their target, if cerebellar granule cells are aded, they form
functional synapses relatively quickly because the target neuron is ther and give the necessary content to form
synapses.

Using a time-lapse video photography, the pontine nuclei extending projections in form of filopodia to seek out an
apropiate contact binding partner.

Intercellular trans-synaptic signalling is also important for excitatory synapse formation,it is possible to demostrate
the presence of neurexin in the cells of pontine nuclei and in presynaptic connections with granule cells dendrites,

Nurexin has a binding partner called neuroligin, which is needed for completing the synapsis process, it acts as an
indicator of the propper cell being reached. It is possible to make them form synapsis with non.neuronal cells using
HEK293, when HEK293 is not transfected axons fibers just grow over surface.

➢ Fibroblast growth factor 22.--> Help in signaling in the pontine nucleus.

SINAPTIC MADURATION

In terms of maturing, once contact is formed, there is a rapid requitement of the needed components for synaptic
transmission. Using cultured neurons which have been transfected with fusion proteins that are able to fluoresce,
ona of the particular snare proteins is marked with a fluorescent marker and it is possible to follow that protein
and observe where it will be visually expressed.

Possible to observe the movement of individual targets. Possible to follow protein release locations and the
translocations of proteins, possible to follow the movement of individual target proteins. Overtime is possible to
observe even translocation of the release sites for those proteins, spotting new releasing sites, this is possible to
prove using particular set of cultures and markers for pre- and post- synaptic locations associated with fluorescent
proteins→ proteins actually migrate to potential synaptic sites, this happens either with pre and post synaptic
proteins.
In terms of what helps to drive the process of synaptic proteins finding special specific sites and forming a cluster,
there is evidence of a ephrin-eph system acting, this system is crucial for receptor clustering. It is possible to show
the labeling of the EphB2 receptor with NMDA-R and taking untreated hypoccampus neurons, which in control
situation have a broad distribution, if the Eph receptors are activated they tend to cluster and form locations→
strong interactions between Eph receptors and NMDA receptors.

Knocking out Eph receptors the clustering gets disrupted.

Functionally, the post synaptic receptors are present early in development before synaptogenesis→glutamate and
recording the sorts of postsynaptic current that is generated→ uniform distribution of response in the whole neuron
with no clustering at all. After synaptogenesis→ hot-spot of glutamate response. This process happens in presence
of TTX so its not dependent on sodium channel→ intercellular signaling driving the process through contact and
interaction.