ADULT AND INFANT

Neurogenesis

Ins Cordn & Ingrid Meucci

Hello everyone,today Ins and I are going to talk about neurogenesis. 30 years from now everyone thought that neurons were formed exclusively during the embryonic development and that the only possible change during adult life was cell death and loss of neurons. But some researchers showed in the early 80s that some parts of the brain may function as germinal centers even in adulthood, as they have the capacity to produce new cells which evolve to new neurons and occupy already functional structures. This formation of new neurons is called neurogenesis

What is Neurogenesis ?
Proliferation & Migration Stem Cell Progenitor Cells

New neurons generated

from neural stem cells and progenitors cells

Oligodendrocyte

Astrocyte

Neuron

Neurogenesis, is not only the generation of new neurons but also other different kind of neuronal cells from the stem cells. Other than neurons, these stem cells produce oligodencrocytes and astrocytes in the brain. The stem cell will rst produce the progenitor cells which will proliferate and migrate, and during this process the genetic and environmental factors will induce this progenitor to produce a specic kind of neuronal cell.

Where does Neurogenesis

Lateral Ventricle Hippocampus

Until now, we know two structures that present neurogenesis during adulthood. First there is the hippocampus, which generates new cells involved in mechanisms such as memorization, and second, there is the subventricular zone, a large germinal region located in the wall of the lateral ventricle.

The SVZ : SubVentricular

In Adult

Cells from this wall have been isolated, and it has been proven that they have the capacity to renew and differentiate into any of the three neuronal type of cells mentioned before. The wall of this subventricular zone is formed by three different layers. First, we have the epithelial layer in contact with the liquid into the lateral ventricle. Next to these cells we nd an hypocellular gap, and then there is a layer of astrocytes in close contact with many blood vessels.

The RMS : Rostral

As we said before, a very important characteristic of these stem cells is the capacity they have to migrate to some specic target regions. It has been found a group of cells that migrates tangentially towards the OB from the SVZ forming a pathway called the rostral migratory stream.

Evidence of Migration in

This route for migration is evident in rodent during infancy and adult life,as you can see in this video

DOES NEUROGENESIS DISAPPEAR ?

It is already widely accepted that neurogenesis takes place in rodents during all their life, but when it comes to humans, this is not so clear. The scientic community agrees with the idea that there is a large neurogenic process during the human embrionic states, but controversy arises when it comes to childhood and adulthood. While some scientics think that this neurogenesis stops just after birth, others think that there is a period of many months, until 18 more or less, were this neuronal process its maintained. In this article, the authors support this second theory were neurogenesis is kept for some months after birth to be then dramaticly reduced until almost disappear during the rest of the adulthood.

Technique:
Stains Mark DAPI All Cells

DCX : Doublecortin

Immature neuron

PSA-NCAM

Migrating neuron

Ki67

Cellular proliferation

GFAP/Vimentine

Astrocytes/Glia

To prove this hypothesis, they used almost exclusively the immunohistochemical techniques. In particular they used these labeling markers DAPI : 4',6'-diamino-2-phenylindole. Binds strongly to A-T rich regions in DNA. DCX : Doublecortin. Neural migration protein expressed by neuronal precursor cells and immature neurons. PSA-NCAM : Polysialyted (PSA) embryonic form of the cell adhesion molecule NCAM. Marks migrating and immature neuron Vimentine : type III intermediate lament protein. Immature glial marker. Ki67 : nuclear protein necessary for cellular proliferation GFAP : glial brillary acidic protein in an intermediate lament. Help to maintain astrocyte mechanical strenght. TH : Tyrosine hydroxylase convert L-Tyrosine to L-DOPA. Source of ROS : Cellular death Calretinin : involved in calcium signaling. Marks interneuron.

Neurogenesis in :
Lateral ventricle

1 Subventricular Zone
Neuroblasts

Rostral Migratory Stream

Olfactory Bulb

3 Olfactory Tract

the hypothesis is that neuroblast from the subventricular zone migrate through the rostral migratory stream to reach the Olfactory bulb. (explication de la gure) In the rst part we will see if there are immature and migratory neurons after birth in the subventricular zone, and then if the rostral migratory stream really exist and what is its composition. And to nish if we nd immature and migratory neurons in the olfactory bulb.

1. Neurogenesis in the SVZ

so to begin, what is happening in the subventricular zone ?

Horizontal Section of the SVZ

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

First, what the authors did, was to proceed doing many horizontal sections of the SVZ at different age stages.

Immunochemistry of the SVZ

During the rst 18 months of age Staining
Cells Immature Neuron Glia

Large number of DCX(+) neurons populate the future gap layer

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

With these horizontal cuts, they did an immunohistochimistry to nd which cells can we nd in the SVZ. This is what they got. In blue we can see the DAPI staining, which marks for the cell density of the area. Then we have the red marker which stains the immature neuron or precursors. And nally we have the green labelling which show us the glia. As you can see, during the rst 6 months after birth there is a dense area next to the ventricular wall, which correspond to immature neuron, shown by the dcx marker. After this 6 month period we see how this neuroblast layer disappears. If we check now the glia (green marker) we appreciate the opposite order of events. During the rst 6 months we cannot see any special event, but later we can see how a dense layer of astrocytes appears. in conclusion during the rst 6 months of life there is a large number of DCX( ) neurons which populate the future gap layer. And then the glial cells will ll in the gap area. After this immunochimistry, they quantifye the number of immature and proliferating neurons. With a DCX stain, they can remark that there is a decline in the number of immature neuron from birth to 9 months. And after 9 months, the number of cells did not change anymore. Its the same for proliferating cells marked with Ki67. So there is a sharp decline of proliferating and immature cells during the rst months of life.

Quantication of Immature

Staining
Immature neuron Proliferating subependymal cell

Sharp decline in proliferating cells and immature neuron

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

This graph shows us how the decrease in the number of immature neurons doesnt only happen until the 18th month but it keeps like this during all adulthood. Ki67 stains proliferating cells, and as you can see they go through the same decrease as the immature neurons. Taken together, these data demonstrate a sharp decline in proliferating cells and immature migrating neurons coinciding with the emergence of the human SVZ gap layer.

Conclusion
During infant and adult age Infant Adult

Where these cells go ?

In conclusion what they saw is a dense layer of immature neurons present in infants that disappeared after 12 months after birth. Now the question was...whats happening with these immature neurons? are they dying? where are they migrating? So the next step was to look at what was happening in the rostral migrating stream.

2. Neurogenesis in the RMS

Caracterization of the RMS

At 5 months Sagital and Coronal Reconstitution
Tributaries of cells Isolated ependymal islets

Evidence of the RMS between the SVZ and the Olfactory Bulb (OB)

Out of the sagital plane

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

When they looked at the RMS what they saw is exactly the expected. With a series of sagital and coronal sections reconstitution they showed how a chain of immature neurons was formed from the SVZ until the OB, demostrating the existence of this migrating stream at 5 months of age. We can appreciate how the proximal area of the RMS is formed thanks to some groups of cells that come from the SVZ.

Immunohistochimie of neurons
At 5 months Staining
Immature neuron Migrating neuron Tributaries of cells Isolated ependymal islets

Clusters of immature and migrating

Out of the sagital plane

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

Once again, to prove that this cells were exactly immature migrating neurons and not any other kind of neurons they used the immunohistochemical technique. And as you can see in the enlargement of this central segment, we can appreciate how cells are dcx and psa-ncam positive cells in this area

Immunohistochimie and
At 1 week
Immature neuron Glia Individual DCX(+) neuronal chains

Label
A : Immature neuron B : Glia Ultrastructural analysis of a neuronal chain cross-section

Evidence of a chain of immature and migrating neurons

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

In these gures we can see how these immature neurons are present in the RMS at 1 week of age and how they are surrounded by astrocytes and often migrate along blood vessels.

General Structure of the RMS

Here we can see a general view of whats happening in an active RMS. Immature neurons coming from the SVZ forming a chain that migrates along the blood vessels towards the OB surrounded by the glia. This migration stream seems to disappear after 18 months after birth but some data has shown how some single or pairs of cells may still be present in older children and adults.

Serial Coronal Sections of the

Staining
Cells Glia

Zhengang Yang et al, Cell Research (2011) 21:1534-1550

First, what the authors did, was to proceed doing many horizontal sections of the SVZ at different age stages.

Horizontal Section of the Adult

Staining
Glia Immature neuron Cells

The RMS-like pathway exists in the adult human

Zhengang Yang et al, Cell Research (2011) 21:1534-1550

First, what the authors did, was to proceed doing many horizontal sections of the SVZ at different age stages.

3. Neurogenesis in the Olfactory Tract

As a nal step, the authors of the article analysed the oltactory tract.

Immunohistochemistry in the
At 1 day,8 months, 7 years Cross-Sectional Reconstitutions
Immature neuron Migrating and immature neuron

Staining
Cells

Decline of an active RMS with age

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

As we can see in the high magnication elds i, j and k, there is a progressive loss of immature migrating neurons also in the olfactory tract. In i there is a high concentration of red and green spots corresponding to these dcx psa-ncam positive neurons, spots that gradually disappear with age to leave only the blue spots corresponding to the DAPI staining at 7 years of age.

Toluidine Blue Staining in a

Central chain of immature neurons surrounded by a matrix of astrocytes

Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

Here we can see a toluidine blue staining of a longitudinal section from a 6 month OT which shows a central chain of darkened immature neurons surrounded by a matrix of lighter-coloured astrocytes.

Quantication of cells in the

At 6 months Proximal RMS

Distal RMS

There is a decline in the total amount of cells and in immature neurons between the proximal and distal RMS
Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

Tracing the infant RMS with serial coronal reconstructions the authors noticed a decrease in caliber of the RMS from the proximal to the distal limb. Based on quantication of DAPI nuclear staining they calculated a decline of a 58% in total cells and a 61% decline in immature neurons. They thought this could be due to immature migratory neurons taking alternative paths, so they kept doing serial coronal reconstructions and.

Hypothesis for decline in cell

Another way of neurogenesis Staining
Immature neuron Migrating and immature neuron

A medial migratory stream (MMS) of immature neurons branches from the proximal rostral migratory
Arturo Alvarez- Buylla et al, Nature. ; 478(7369): 382386. doi:10.1038/nature10487

what they found is exactly that. A second migration stream called Medial Migration Stream (MMS) that branched off the proximal limb of the RMS and goes to the ventro-medial pre-frontal cortex (VMPFC). This MS was observed in human specimens of 4-6 months but not in 8-18 months specimens. As in the RMS, they repeted the dcx and psa-ncam staining to demostrate they were immature migrating neurons, and as you can see in gures f and e the cells were dcx and psa-ncam positive.

Summary
Lateral ventricle Subventricular Zone Neuroblasts Rostral Migratory Stream Olfactory Bulb Olfactory Tract

Why RMS is no longer active in

Other pathways like MMS Serial sections (sagital, coronal, horizontal) : DAPI If yes : Immunohistochemistry (DCX, Hud+/Sox10-, NeuN) Apoptosis ? Hybridation in-situ : caspase, P53 ? Which are the transcription factors expressed by the astrocytes ? Transcriptome : microarray