MESSY FIBER GROWTH AND SYNAPTOGENESIS IN RAT HIPPOCAMPAL SLICES IN-VITRO

Hippocampal slices from early postnatal rat were used to study messy f iber (MF) growth and synaptogenesis. The ability of MFs to form new gi ant synapses within isolated tissue slices was established by a series of experiments involving synapsin I immunohistochemistry, electron mi croscopy, and whole-cell recordings. When hippocampal slices from imma ture rats were cultured for up to 2 weeks, the distribution of giant M F terminals was similar to that found in vivo. Using a lesioning proce dure, we determined that MFs in slices extend and form appropriate syn aptic connections with normal target CA3 pyramidal cells. MF terminals were dispersed more widely than normal within the CA3 pyramidal layer after a lesion, but electron microscopy indicated that synaptic junct ions were still primarily associated with pyramidal cell dendrites and not the somata. Establishment of functional synaptic input in vitro w as confirmed by whole-cell recordings of MF-driven excitatory postsyna ptic currents (50 pA to 1 nA) in pyramidal cells. The results establis h for the first time that an MF projection with appropriate and functi onal synaptic connections can be formed de novo and not just maintaine d in excised hippocampal slices. The cellular dynamics underlying MF g rowth and synaptogenesis were examined directly by time-lapse confocal imaging of fibers selectively stained with a fluorescent membrane dye (Dil or DiO). MFs growing deep within isolated tissue slices were tip ped by small (5-10 mu m), active growth cones that advanced at variabl e rates (5-25 mu m/hr). Furthermore, dynamic filopodial structures wer e seen at small varicosities along the length of developing MFs, which may identify nascent on passant synaptic contacts. The hippocampal sl ice preparations are shown to support normal development of Mf connect ions and allow for direct visualization of the cellular dynamics of sy napse formation in a mammalian CNS tissue environment.