![\"SJLab2\"](\"http:\/\/ventricular.org\/StephenNoctor\/wp-content\/uploads\/2015\/11\/SJLab2.jpg\")
<\/a><\/p>\nMy graduate thesis studies focused\u00a0on histogenesis of ferret somatosensory cortex. Several facets of ferret brain structure and development are unique in comparison to other animal\u00a0models often used in developmental studies. For example, the ferret cerebral cortex is gyrencephalic, unlike the smooth lissencephalic cortex of mice and rats.<\/p>\n
<\/a>Photo courtesy of Sharon Juliano and Krys Rodomski<\/p><\/div>\n
If you haven’t seen a ferret brain, the image to the right shows\u00a0the degree of gyrification, or folding, of ferret cerebral cortex. In addition,\u00a0cortical neurogenesis\u00a0continues after birth in ferrets, while\u00a0this process is complete by the time of birth\u00a0in most mammals. Furthermore,\u00a0while cortical neurogenesis occurs over a period of about 7 days in mice and rats, the neurogenic period of ferret cortical development is much longer, occuring over 4+ weeks. These differences provide distinct advantages for teasing apart factors that guide developmental processes in the forebrain.<\/p>\n
Jackson, Peduzzi and Hickey published a thorough\u00a0paper in 1989 detailing the temporal gradients of\u00a0neurogenesis and neuronal migration in ferret visual cortex. Sharon, Nate and I published a\u00a0paper in 1997<\/a><\/em><\/span> that determined\u00a0when each layer of the cerebral cortex was\u00a0generated in ferret primary somatosensory\u00a0cortex. With that knowledge in hand, we used MAM to interfere with the production\u00a0of specific cortical layers in\u00a0ferret somatosensory cortex.<\/p>\n MAM (Methylazoxymethanol acetate) is a neurotoxin that reduces DNA synthesis by methylating DNA and has a half-life of about 12 hour at physiological\u00a0pH. Our goal was to use MAM\u00a0to temporarily stop cell division in the cortical proliferative zones during production of neurons\u00a0destined for\u00a0specific\u00a0cortical layers. Since ferret cortical neurogenesis is protracted, periods of at least three days can be identified when neurons destined for a single cortical\u00a0layer are being generated. It is therefore possible to target \u00a0individual layers.<\/p>\n MAM treatment on E34 disrupted production of layer 4 in somatosensory cortex,\u00a0significantly reducing the thickness and the number of cells in layer 4. Not surprisingly, when Sharon, Sid, and Deb\u00a0looked at adult animals they\u00a0found that E34 MAM treatment altered the distribution of cortico-thalamic terminations, as well as the timing and\u00a0flow of sensory information into somatosensory\u00a0cortex and between\u00a0cortical layers.<\/p>\n We looked more closely at radial glial morphology by preparing acute slice cultures of embryonic ferret cortex obtained from control and experimental animals. We placed discrete injections of fluorescent tracers into the ventricular zone and kept the slices alive long enough for the \u00a0fluorescent dye\u00a0to fill labeled cells.<\/p>\n MAM treatment at E34 reduced the number of layer 4 neurons, but left many features of cortical organization intact. In contrast,\u00a0MAM treatment\u00a0targeting\u00a0earlier stages of cortical development resulted in profound disruption. Very thin cortical grey matter, multiple ectopic clusters of cortical neurons with abnormal orientation, Cajal Retzius cells distributed across\u00a0the cortical wall rather than confined\u00a0to\u00a0the marginal zone, and RG cell morphology that was thoroughly altered. RG pial fibers\u00a0were no longer oriented along a radial trajectory, there was a\u00a0precocious\u00a0onset of GFAP expression, and RG cells appeared to\u00a0begin\u00a0translocating earlier than normal.<\/p>\n The scope of these projects provided me with a solid foundation in fundamentals of cortical development, and I am of course forever indebted to Sharon. It also piqued my interest in radial glial cells and their translocation. Enough so that I included the slide below in my thesis talk. \u00a0Yes, I made that slide in Powerpoint…. \u00a0don’t you just love the standard blue background \/ yellow text format? It’s screaming: I’m a child of the 90’s!! The question mark was meant to indicate “what happens\u00a0next?” I’ve written a bit\u00a0more on the story of translocating radial glia – including data from my first publications on\u00a0translocating radial glia in 2004 and 2008 – in the ‘Time-Lapse Movies<\/a><\/em><\/span>‘ section on\u00a0this website. But\u00a0there will be\u00a0more to this story in an upcoming post. Stay tuned!<\/p>\n<\/a>We\u00a0tested whether essential\u00a0programs of cortical development would resume after MAM treatment. The image on the right shows BrdU labeled neurons (the black dots) in the adult ferret somatosensory cortex after MAM treatment. This animal received MAM treatment on E34, when layer 4 neurons were being produced, and a single BrdU treatment three days later when layer 3 neurons were being produced. This experiment demonstrated that neurogenesis resumed after E34 MAM treatment, and that the BrdU-labeled cortical neurons migrated to their appropriate location after the E34 MAM treatment.<\/p>\n<\/a>However, when we examined the neonatal brain, we saw\u00a0that the E34 MAM treatment did not appear to disrupt basic features of developing cortical cytoarchitecture. Despite halting DNA synthesis at E34, the RG cell scaffold retained it’s\u00a0fundamental\u00a0morphology, and based on what we know now, the RG cells also appear to have retained their neuron\u00a0generating\u00a0potential since neurons destined for layers 2 and 3 were produced after the MAM treatment. The image on the right shows vimentin staining of P0 ferret somatosensory cortex in control and E33 MAM treated ferrets. The trajectory and density of radial glial fibers\u00a0was not significantly altered by the\u00a0MAM treatment.<\/p>\n<\/a>These were fun experiments for several reasons. The fluorescently labeled radial glial cells were just absolutely beautiful. The image above on the left was taken on\u00a0a Biorad confocal, and while you can detect some features of the RG cells, it really\u00a0doesn’t quite capture the beauty of seeing these cells in person on the microscope. And because Sharon really enjoyed working at the bench we did these experiments together. She made\u00a0made the injections and my\u00a0job included\u00a0making sure the slices\u00a0were healthy, monitoring CSF flow rate in the slice chamber, fixing the slices, co-staining with various antibodies such as vimentin to test for RG identity, imaging, etc. I’d then \u00a0use Neurolucida to trace the trajectory of the RG pial fibers in control and experimental groups. Panels A and B in the figure above show sample tracings of\u00a0injections that Sharon\u00a0placed in the VZ of control\u00a0and E33 MAM treated animals.<\/p>\n<\/a><\/p>\n