Adult neurogenesis Flashcards
What is the journey of a progenitor to differentiated cell in early development and adulthood
In early development neural stem cells divide symmetrically to generate more neuro-epithelial cells. As the developing brain epithelium thickens, neuroepithelial cells elongate and become radial glial cells. These divide asymmetrically to generate neurons directly or indirectly through intermediate progenitor cells. Oligodendrocytes are also derived from radial glia through intermediate progenitor cells. As the progeny of RG and IPCs move into the mantel for differentiation, the brain thickens, further elongating RG cells. At the end of development most RG begin to detach from the apical ventricle and convert into astrocytes. Neonatal RG continue to generate neurons and oligonucelotides; some convert into epyndymal cells, whereas others will convert into adult SVZ astrocytes that continue to function as NSCs in the adult.
Why might we suspect that a stem cell population persists in the hypothalamus
Hypothalamus controls core body functions - composed largely of a series of interconnected nuclei. Also monitoring of many circulating body signals (hormones metabolites etc) As these change constantly - so do the requirements of the animal through changes in behaviour. Might be useful therefore if the hypothalamus can adapt to anticipate and meet these changing conditions
What part of the hypothalamus was studied in mice
Section cut through the median eminence of the hypothalamus. Specialised radial glia like cells line the ventral half of the medial hypothalamus - hypothalamic tancocytes
What are the characteristics of hypothalamic tancycytes
Look like radial glial cells
Cell body makes basal contact with the ventricular layer of the third ventricle
A single long basal process emerges from the cell body and makes contact with different targets depending on the location of the cell body
What studies were performed to investigate the stem like character of tancycytes
In rat and mouse - De novo neurogenesis was seen in response to long term changes e.g high fat diet. To see if the new born neurons are from tancycytes:
Inject EDU into the mouse third ventricle, this will be incorporated into replicating cells in S phase (EDU is a thymine analogue) Then the mouse is killed and cells are inspected - able to see which cells are new born i.e which cells have taken up the EDU.
How are tissue specific lineage tracing or KO experiments performed
- Lineage tracing - Breed one mouse strain with a reporter gene downstream of a stop codon that is ubiquitously expressed. Also have the Stop codon floxed (Lox P) Combine this strain with one that expresses cre recombinase under the promoter of a specific tissue only. In the presence of Cre recombinase, the Stop codon is excised - reporter gene is expressed only in the specific tissue where the tissue specific promoter is found.
- Condition KO - Cre recombinase under tissue specific promtoer
Flox an exon required for targeted protein expression. Mouse strain hybrid = KO of protein in specific cells, only where the cre tissue specific promoter is found.
How can temporal conditional techniques be performed
Use CreERT2 which encodes a Cre recombinase fused to an oestrogen ligand-binding domain that requires the presence of tamoxifen to become active. This means Cre recombinase is only active following injection of tamoxifen into the mouse.
What was the temporal conditional technique used to study tancycytes
In tancycytes GLAST is a stem cell specific marker
CreERT2 was placed underneath GLAST promoter
Floxed stop codon under promoter ROSA26, also placed LacZ under STOP codon. After tamoxifen addition, Beta galactosidase expression was seen in the alpha tancycytes and absent ventrally in beta tancycytes.
Why were the mice used in the temporal conditional technique killed immediately first time around
Check that the only cells to incorporate the reporter gene are the tancycytes. i.e no neurons will be expressing GFP. Repeat and let the mice survive for much longer.
What were the results of leaving the mice to survive for 9 months after tamoxifen addition
Alpha tancycytes were shown to self renew, give rise to other tancycyte subsets, to neurons and to astrocytes
What can be said about the rate of proliferation, neurogenesis and gliogenesis of tancycytes in normal caged conditions
Very low (low stress)
What was the complementary in vivo study performed to show that tancycytes exhibit neural stem cell properties
1) Dissection of hypothalamic region
2) enzymatic dissociation (trypsin)
3) Mechanical dissociation of one cell per tube
4) Plate in suspension culture with growth factors
5) incubate for 2 weeks
This produced a neurosphere
What was done to the produced neurosphere in order to test its stem cell like character
1) put it into differentiating conditions - this produced arcuate neurons
2) Re dissociate the neurosphere to a single cell and see if it reforms a neurosphere - it does
What evidence is there that the alpha tancycytes derive from FGF10+ embryonic multipotent hypothalamic progenitors.
They themselves express FGF10 - after addition of more FGF10 more proliferation in these cells was seen.
What is the Hes5:CreERT2 mouse currently being used to investigate
Whether corticosterone stimulates tancycytes in vivo.
