POLARIZED DISTRIBUTION OF THE TRANS-GOLGI NETWORK MARKER TGN38 DURING THE IN-VITRO DEVELOPMENT OF NEOCORTICAL NEURONS - EFFECTS OF NOCODAZOLE AND BREFELDIN-A

Neurons are polarized secretory cells whose cytoplasm and plasma membrane are polarized to form two compartments: dendrites and axons. In mature, fully polarized neurons, the microtubule-associated protein Map2 is targeted to dendrites, while tau is mainly restricted to axons. However, the intraneuronal distribution of secretory pathway organelles, such as the endoplasmic reticulum and the Golgi complex, which give rise to all constitutive regulated and lysosome vesicles, is poorly understood. Thus, to investigate the distribution of the trans-Golgi network during the development and maturation of rat neocortical neurons in vitro, we have utilized an antibody recognizing a 38 kDa trans-Golgi network-specific protein, TGN38, and immunofluorescence microscopy. Before neurons have established polarity, TGN38 immunoreactivity outlines several vesicles dispersed throughout the cell body cytoplasm; these converge close to a major Map2-immunopositive process during the establishment of neuronal polarity, and later merge into a single structure located at the base of a thick Map2-immunopositive process, similar to 18 h after plating. At this stage TGN38 immunoreactivity is located within 45 degrees of the major Map2-immunoreactive process in 54% of neurons, while in only 6% of cells it is located at the opposite pole. After 3 days in vitro, during the segregation of microtubule-associated proteins to either dendrites or axons, TGN38 immunoreactivity clusters continue to be located close to a major dendrite, and in some neurons these clusters begin to enter a major Map2-immunoreactive process. At 10 days in vitro TGN38 immunoreactivity extends into a major dendrite for 5 - 30 mu m in many neurons. Thus, the distribution of TGN38 immunoreactivity becomes polarized, being localized within a single, usually the major, neocortical dendrite. Our results also show that the morphological appearance of TGN38-immunoreactive structures is microtubule-dependent, since nocodazole treatment of polarized neurons induces scattering of TGN38-immunoreactive vesicles throughout the cell body's cytoplasm. Treatment with brefeldin A induces scattering of small TGN38-immunoreactive vesicles throughout the neuronal cytoplasm and processes, a different response to that observed in non-neuronal cells.