Question

You have discovered a new species of platypus with an unprecedented 7 layer cortex, with an additional neuronal layer compared to the typical 6 layers mammalian cortex. Propose an experiment to determine in what sequence the neuron layers were generated during brain development in this platypus.

This question has been written based on the following article:

" The determination of neuronal fate in the cerebral cortex", Review article by Susan K. McConnell

Answer 1

Ans: Early age stimulation, or how we nurture a given animal/mammal leads to better cerebral cortex development. Susan K. McConnell studied the fate of cortical neurons by isochronic and heterochronic transplantation of early stage progenitor cells

Exp1: Normal development of neurons in the primary visual cortex (area 17) of the ferret.

Injections of 3H-thymidine into newborn ferrets showed that neurons generated after birth are destined to sit in layer 2/3 of the cortex, whereas neurons born on embryonic day (E) 32 populate primarily layers 5 and 6. There is a chance that day (E) 32 neurons populate additional layer 7

Many layer 2/3 neurons in adult ferrets could be retrogradely labeled with HRP from visual cortical areas 16 and 19, while about half of the neurons in layer 6 were found to project to the lateral geniculate nucleus (LGN).

Exp2: presumptive layer 2/3 cells were labeled in vivo by injecting ferrets with 3H-thymidine on PI and P2. Before the cells had a chance to migrate, they were removed from the donor brain, incubated in a fluorescent dye (DAPI or fast blue), and dissociated into a single-cell suspension. The labeled cells were then transplanted into the proliferative zone of a littermate host ferret (“isochronic” transplants). Over the next few weeks, many of these dye-labeled cells underwent changes in their position and morphology that were consistent with a radially directed migration and subsequent differentiation into cortical neurons. The final positions of isochronically transplanted neurons in the host brain were mapped out by using the 3H-thymidine marker after long survival periods. About 97% of radioactively labeled cells had migrated out into the visual cortex, where they attained a compact laminar distribution: 99% were found in layer 2/3, their normal destination. The labeled cells had normal, mostly pyramidal neuronal morphologies and appeared to be well integrated with host neurons when viewed in Nissl-stained sections. Ten isochronically transplanted neurons were successfully labeled after HRP injection into 2 normal target regions, areas 16 and 19. Thus, newly generated cortical neurons are capable of survival, migration, and differentiation in a host brain, and transplantation per se does not alter their fate.

Exp 3: In a final set of experiments, the commitment of presumptive deep-layer neurons to their normal fate was tested by challenging them to alter that fate. In these “heterochronic” transplants, cells from the occipital proliferative zone of E31 or 32 donor ferrets were labeled with 3H-thymidine and then transplanted into the proliferative zone of a newborn host ferret. In contrast to the isochronic transplants, about 60% of 3H-thymidine-labeled cells in heterochronic transplants failed to migrate and were instead found at the site where they were injected. Here there is a chance that more percentage of cells might have migrated to cortex area to make 7^th layer.

The remainder migrated out to the visual cortex and developed neuronal morphologies. Of the labeled cells that reached the cortex, >43% were found in layer 2/3, and the remaining >57% occupied the deeper cortical layers, primarily layers 5 and 6 their normal destination but due to excess cells entering cortex area might have formed 7^th layer of cortex. Five transplanted neurons sitting in layer 6 were retrogradely labeled from the LGN, a normal target of layer 6 cells. These results indicate that at least a subpopulation of embryonically generated neurons appears to be committed to a deeplayer fate prior to migration.