BackgroundHigher mammals such as primates and carnivores have highly developed unique brain structures such as the ocular dominance columns in the visual cortex, and the gyrus and outer subventricular zone of the cerebral cortex. However, our molecular understanding of the formation, function and diseases of these structures is still limited, mainly because genetic manipulations that can be applied to higher mammals are still poorly available.ResultsHere we developed and validated a rapid and efficient technique that enables genetic manipulations in the brain of gyrencephalic carnivores using in utero electroporation. Transgene-expressing ferret babies were obtained within a few weeks after electroporation. GFP expression was detectable in the embryo and was observed at least 2 months after birth. Our technique was useful for expressing transgenes in both superficial and deep cortical neurons, and for examining the dendritic morphologies and axonal trajectories of GFP-expressing neurons in ferrets. Furthermore, multiple genes were efficiently co-expressed in the same neurons.ConclusionOur method promises to be a powerful tool for investigating the fundamental mechanisms underlying the development, function and pathophysiology of brain structures which are unique to higher mammals.

Elucidating neuronal circuits and their plasticity in the cerebral cortex is one of the important questions in neuroscience research. Here we report novel axonal trajectories and their plasticity in the mouse somatosensory barrel cortex. We selectively visualized layer 2/3 neurons using in utero electroporation and examined the axonal trajectories of layer 2/3 neurons. We found that the axons of layer 2/3 neurons preferentially run in the septal regions of layer 4 and named this axonal pattern "barrel nets." The intensity of green fluorescent protein in the septal regions was markedly higher compared with that in barrel hollows. Focal in utero electroporation revealed that the axons in barrel nets were indeed derived from layer 2/3 neurons in the barrel cortex. During development, barrel nets became visible at postnatal day 10, which was well after the initial appearance of barrels. When whisker follicles were cauterized within 3 d after birth, the whisker-related pattern of barrel nets was altered, suggesting that cauterization of whisker follicles results in developmental plasticity of barrel nets. Our results uncover the novel axonal trajectories of layer 2/3 neurons with whisker-related patterns and their developmental plasticity in the mouse somatosensory cortex. Barrel nets should be useful for investigating the pattern formation and axonal reorganization of intracortical neuronal circuits.

SummaryBrain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

No abstract