Notes

Neurulation

How do you get from a fertilized egg and pluripotent cells to the highly specialized neural structures?

Principles of Mammalian Neural Development

• Tissue Polarity: The tissue is polarizing.
• Cellular Polarity: also cells have to be polarized.

Neurulation It is the start of the formation of the CNS. It is the formation of the vertebrate nervous system in embryos. The notochord induces the formation of the CNS by signaling the ectoderm above it to form the thick and flat neural plate. The neural plate then folds in on itself to form the neural tube, which will then later differentiate into the spinal cord and brain. In the figure: (top) Neuro-epithelial layer already showing signs of elongation of cells (notochord not present). (middle) Neural groove forming. (bottom) Neural tube formed with overlying ectoderm and underlying notochord.

How to visualize dividing cells (and their progeny)? Dividing cells like stem cells are those that give rise to neurons. To visualize them we can use:

• Endogenous marker (pH3. Ki67, mitotic figures): are proteins that selectively express when the cell is dividing.
• Thymidine analogues (BrdU): these analogues are integrated into the DNA when it is doubling and they can be detected through antibodies.
• Retroviruses: they cannot cross nuclear membrane, so they can only enter the cell when it is dividing and can be later identified.
• Transgenic Lineage Tracers
• Fusion Plasmids (tubulin, histones).

Transgenic Fate Tracing

Radial Glia as Neural Stem Cells? How could you identify if these cells give rise to neurons within the developing cortex. It is done through Transgenic Fate Tracing.

Transgenic Fate Tracing It is a method used in developmental biology to study the development and differentiation of cells in an organism. It involves creating genetically modified organisms that express a certain gene or set of genes specifically in certain cell types of interest. The gene used for fate tracing are typically linked to a fluorescence or other type of reporter molecule, which allows the cells of interest to be visualized and tracked over time. The basic steps of transgenic fate tracing are:

• Identification of a gene that is specifically expressed in the cell type of interest.
• Creation of a transgenic organism that expresses the gene in the cells of interest.
• Observing the expression of the gene and the development and differentiation of the cells in the organisms. By tracking the cells that express the fate-tracing gene, researchers can gain insights into the origins and developmental pathways of different cell types.

In Malatesta 2003, it is done through Cre-recombinase, which are bacterial enzymes normally not present in the body that can cut DNA or recognize specific DNA patterns (loxp). You express this Cre-recombinase under GFAP (highly expressed in Glia cells), thus now driving also Cre-recombinase. The researchers generated transgenic mice that expressed a green fluorescent protein (h-GFAP) under the control of a promoter that was specifically active in neuronal precursors. Using these transgenic mice, the authors were able to visualize and track the development and differentiation of neuronal precursors in the hippocampus in real-time, and to demonstrate that these cells gave rise to both neurons and astrocytes.

Retroviruses Moloney murine leukemia (MML) viruses cannot cross nuclear membrane: specific for dividing cells. Then, when the cell divides, the retrovirus can enter the nuclear membrane and fluorescent protein (GFP) can be used to evidence it (Retroviral Labelling).

Radial Glia Divide and Generate Neuronal Progeny There are two main ways in which Radial Glia divides:

• Asymmetric: in this case, the radial glia cell divides into two daughter cells, one of which retains the characteristic of a radial glia cell and can continue to divide and support neuronal migration, while the other differentiates into a neuron. (Most common scenario).
• Symmetric: it generates a "daughter" which is a basal progenitor that can be divided again and it usually give rise to two neurons.

Hippocampal Adult Neurogenesis

"Once development was ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, and immutable: everything may die, nothing may be regenerated" - Ramon y Cajal, 1928.

Early Evidence for Cell Division in the Adult Brain Before

After

Plasticity and Repair of the Mammalian Brain

• Life-long neurogenesis in the adult hippocampus in mammals including humans. (still largely controversial).
• Involved in certain forms of learning and memory.
• Reduced and/or altered neurogenesis in neuro-psychiatric disease (e.g., depression, ageing).
• Areas involved in neurogenesis in adult human brains: dentate gyrus that is the entrance door to hippocampus, adult rodent brains: subventricular zone that migrate and differentiate in olfactory bulb cells.

In order to identify neural stem cells in the Dentate Gyrus of Hippocampus Intravital Imaging can be used, which allows to very deep within the tissue with a 2-photon technique. tdTOMATO is injected to identify the cells. This technique allows for the construction of a lineage tree of adult neural stem cells deriving from the same mother cell.

One mother and her daughters Things to look at:

• Stem cell dynamics
• Mode of cell division
• Migration and Integration
• Functional Properties (combined with GECIs)
• Self-Renewal Potential There is evidence suggesting long-term self-renewal, but there is also evidence suggesting the contrary, i.e., rather quick depletion.

Distinct Behaviour of Neural Stem Cells in the Hippocampus These two types of cells can be identified through molecular signature.

Diversity of Hippocampal Stem Cells

• Functional and molecular diversity of hippocampal neural stem cells.
• Long-term (>100 days) self-renewing stem cells exist in the adult mammalian hippocampus.
• How do they contribute to life-long neurogenesis?
• What are the stem cell dynamics with advancing age?

However, a main question is the prior history of a given cell and this can be investigated through iCOUNT.

Dynamic Regulation of Adult Neurogenesis The number of neurons generated is not static but dynamically regulated. Elements that induces an increase in the amounts of neurons generated are:

• Voluntary physical exercise doubles the amount of neurons generated in the hippocampus.
• Learning
• Housing of mice in an environmental enrichment, where they live in a much larger cohorts with more mouses.

One experiment demonstrating how enriched environment enhances learning based on Hippocampus is the Morris Water Maze. Indeed, mouses grown in an enriched environment showed much faster learning curves in finding the platform in the water maze compared to mouses grown in less "fancy" environments. Hence, there is some kind of correlation between increased hippocampus neurogenesis and performance in learning spatial locations, even though it has not yet been demonstrated a causality. Other examples, Cab driving increases hippocampal size: hippocampal volume correlates with the time spent behind the wheel. Students learning increases brain size.

There are however also negative regulators of neurogenesis (also involved with neurodegenerative diseases):

• Alcohol
• Stress: if you stress mice it dramatically reduces the amount of neurons generated in the hippocampus, which in turn has control over mood and can be implicated in depression. Indeed, studies demonstrated that antidepressant treatment are correlated with increased neurogenesis in adult rat hippocampus. Further studies, showed that there has to be neurogenesis happening in the hippocampus for antidepressants to work.
• Age
• Hit-Damage.

Irradiation Ablated Neurogenesis By using ionizing radiation, which divides cells (like those used in cancer therapy), dramatically reduces neurogenesis in the HC. In Santarelli et al. 2003, they studied mice that were treated with ionizing radiation to ablate neurogenesis in the hippocampus and then administered the antidepressant fluoxetine (Prozac). The results showed that ablation of neurogenesis in the hippocampus prevented the behavioral effects of fluoxetine, suggesting that neurogenesis in the hippocampus is required for the antidepressant effects of this drug.

Other approaches to ablate neurogenesis involve transgenic mice (using TK or suicide genes under the control of stem cell promoters) and local cell ablation (e.g., lentiviral vectors). All of these approaches are not perfect and present disadvantages, hence they are often used in combination to mitigate cons.

The Dentate Gyrus as a Pattern Separator The Dentate Gyrus represents very similar inputs distinctly within the brain. What it has been shown is that mice with decreased neurogenesis are impaired at spatial pattern separation.