Cerebellar organotypic cultures

Ji F, Obata K (1999) Development of the GABA system in organotypic culture of hippocampal and cerebellar slices from a 67-kDa isoform of glutamic add decarboxylase (GAD67)-deficient mice. Neurosci Res 33 233-237. 

Fig. 7. Living nerve cells in organotypic culture viewed with phase-contrast microscopy. (A) Cerebellar Purkinje cells, 38 days in vitro. (B) Pyramidal cells in thin area of a hippocampal culture prepared from a newborn rat. (C) Layer of pyramidal cells in hippocampal culture prepared from 8-day-old rat. (D) Large nerve cells of the medulla. Scale, 30 pm. (From Gahwiler, 1980.)...

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

In this chapter we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar primary cultures and organotypic slices. 

One of the main advantages from the use of organotypic cultures (Fig. 3a) is that this type of preparation allows maintaining an intact or semi-intact architecture of the tissue, so that, for example, transfected cells are immediately spotted at low magnification and their locations can be easily tracked (Fig. 3b). If a fluorescent nuclear stain (DAPI) is applied, it is also possible— in our example— to map with good precision the individual layers of the cerebellar cortex (Fig. 3c), and/or to label live/dead cells with two color fluorescence (Fig. 3d). 

Hyperbranched PDMAEMA Rat organotypic brain slice culture 48 h 1-10 pg Cortical and cerebellar slices 44... 

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