THE GOLGI-APPARATUS AND THE CENTROSOME ARE LOCALIZED TO THE SITES OF NEWLY EMERGING AXONS IN CEREBELLAR GRANULE NEURONS IN-VITRO

Cultured cerebellar granule neurons develop their characteristic axona l and dendritic morphologies in a series of discrete temporal steps hi ghly similar to those observed in situ, initially extending a single p rocess, followed by the extension of a second process from the opposit e pole of the cell, both of which develop into axons to generate a bip olar morphology. A mature morphology is attained following the outgrow th of multiple, short dendrites [Powell et al., 1997: J. Neurobiol. 32 :223-236]. To determine the relationship between the localization of t he Golgi apparatus, the site of microtubule nucleation (the centrosome ), and the sites of initial and secondary axonal extension, the intrac ellular positioning of the Golgi and centrosome was observed during th e differentiation of postnatal mouse granule neurons in vitro. The Gol gi was labeled using the fluorescent lipid analogue, C-5-DMB-Ceramide, or by indirect immunofluorescence using antibodies against the Golgi resident protein, alpha-mannosidase II. At 1-2 days in vitro (DIV), th e Golgi was positioned at the base of the initial process in 99% of un ipolar cells observed. By 3 DIV, many cells began the transition to a bipolar morphology by extending a short neurite from the pole of the c ell opposite to the initial process. The Golgi was observed at this si te of secondary outgrowth in 92% of these ''transitional'' cells, sugg esting that the Golgi was repositioned from the base of the initial pr ocess to the site of secondary neurite outgrowth. As the second proces s elongated and the cells proceeded to the bipolar stage of developmen t, or at later stages when distinct axonal and somatodendritic domains had been established, the Golgi was not consistently positioned at th e base of either axons or dendrites, and was most often found at sites on the plasma membrane from which no processes originated. To determi ne the location of the centrosome in relation to the Golgi during deve lopment, granule neurons were labeled with antibodies against gamma-tu bulin and optically sectioned using confocal microscopy. The centrosom e was consistently co-localized with the Golgi during all stages of di fferentiation, and also appeared to be repositioned to the base of the newly emerging axon during the transition from a unipolar to a bipola r morphology. These findings indicate that during the early stages of granule cell axonal outgrowth, the Golgi-centrosome is positioned at t he base of the initial axon and is then repositioned to the base of th e newly emerging secondary axon. Such an intracellular reorientation o f these organelles may be important in maintaining the characteristic developmental pattern of granule neurons by establishing the polarized microtubule network and the directed flow of membranous vesicles requ ired for initial axonal elaboration. (C) 1998 Wiley-Liss, Inc.