Confocal imaging of mossy fiber growth in live hippocampal slices.

Acutely isolated slices of developing rat hippocampus have been used to study axon growth and synapse formation. Mossy fibers, which are the axons of dentate granule cells, were labeled in living brain slices by injection of a fluorescent membrane dye (DiI or DiO) into the dentate gyrus. Time-lapse observations were made in area CA3 at a time when mossy fibers are normally growing in and forming en passant synapses with pyramidal neurons. Single scan images were collected at 1-2 min intervals over a period of several hours using a scanning laser confocal microscope. At the tips of growing mossy fibers were highly motile growth cones with several filopodia and small lamellae. Labeled fibers typically extended at rates up to 15 microns/h, but occasionally individual axons abruptly stopped elongating and the leading growth cone became quiescent. In addition, dynamic filopodia-like structures were found to be associated with axonal varicosities proximal to the leading growth cone. We are currently pursuing methods to determine whether these motile activities correlate with synapse formation.