Question

1. A) What is “Brainbow”? How does the system work? If you wanted to understand the neuronal connections that are responsible for coordinating voluntary motor function which cell type would you label with brainbow? What specific transgenes would you use? Paste a picture of the cell type labeled with brainbow in your response.

B) What is BrdU staining? What information does it give you about cells? How can it be used to identify the timing of neuron birth in relation to development of the neocortex?

Answer 1

1) BRAINBOW

Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of connectomics, traditionally known as hodology, which is the study of neural connections in the brain.

Brainbow is a genetic cell-labeling technique where hundreds of different hues can be generated by stochastic and combinatorial expression of a few spectrally distinct fluorescent proteins.

B)BRDU STAINING:

BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells. Print this protocol. BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal.

BrdU is commonly used in the detection of proliferating cells in living tissues. 5-Bromodeoxycytidine is deaminated to form BrdU.

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form “isochronic clusters.” Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.