Organotypic slices in vitro: repeated,same-cell,high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live,transfected cerebellar neurons by confocal microscopy

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports. This system permits cultures to be repeatedly observed both by phase contrast microscopy and, upon biolistic transfection, by laser confocal microscopy using 40x, 60x, and 100x water-immersion objectives, at high resolution. Upon co-transfection with two plasmids, differentiation of the same transfected Purkinje neurons was followed across in vitro development for periods of up to 10 days. Despite the relative thickness of the slice culture, even small, punctate, nuclear signals, were detectable. The results show that Purkinje neurons in cerebellar slices explanted from postnatal day 2 mice, developed cytotypically, although some were ectopically located. In contrast, Purkinje neurons in slices from postnatal day 6 cerebella developed in an organotypic manner. It is concluded that this culture system serves as an ideal tool for applications in experimental biology where high resolution tracking of cellular signals, over extended time periods, is of interest.