Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

[Transgene 6A-99 is a molecular marker of developing somatosensory cortex in mice].

Among dozens of known genes, active in the developing cortex, none possessed expression strictly limited by one functional area of the cortex. We found that in 6A-99 transgenic mice, Lac-Z-reported gene was expressed selectively in the somatosensory cortex. In the primary somatosensory cortex, expression was localized in the barrel field, including the zone of representation of vibrissae, fore and hind limbs, jaws, and head. In addition, LacZ-expressing cells were found in the secondary somatosensory cortex, as well as in the nuclei of hypothalamus and dorsal and caudal raphe nuclei. In the cortex, expression of transgene 6A-99 began on day 3 of postnatal development (P3) and embraced only the area of primary somatosensory cortex: zones of representation of the snout, vibrissae, and lower jaw. On P5, pronounced expression 6A-99 was detected in the secondary somatosensory cortex and zone of representation of fore paws. Expression in the zone of representation of hind paws was observed on P7. Expression of 6A-99 in the somatosensory cortex disappeared by P50, the age of final functional maturation of the cerebral cortex. Our results suggest that the regulation of transcription in the developing mouse somatosensory cortex differs from those in other cortical areas. Transgene 6A-99 may serve as a specific molecular marker of the developing somatosensory cortex in mice and can be used for studying the mechanisms underlying genetic and epigenetic control of the neocortex functional regionalization.