A fluorescent tracing of hilus-granular organization utilizing visual feedback system

The neuronal organization of the hippocampus is extensively studied. However, the synaptic connections between neurons of the hilus and neurons of the granule cell layer are still debatable. In the present study, we utilized automated micromanipulation technique combined with visual image processing to analyze the synaptic connections between hilar mossy cells or mossy fibers, and granule cells or basket cells in the granule cell layer. The stereoscopic microscope image of the dentate gyrus of coronal slice in adult rat brain was converted into a binary image and the noises were reduced by median filter. The threshold for binary image was determined by the mean concentration and standard deviation calculated from the dentate gyrus. The area of the dentate gyrus is extended by region segmentation, and the two-valued image was constructed based on mean concentration and the standard deviation. Noises were further reduced by the dilation-erosion processing. This was matched with the template image made from a microscopic photo of cresyl violet-stained coronal section of the rat brain by parallel and rotation matching. A glass microelectrode was inserted into the apex of the dentate gyrus by the automated micromanipulation technique. Lipophilic fluorescent tracer DiI or DiD was injected into the hilus by a nanoinjector. Successful tracing of hilar neurons was observed by a confocal laser scan microscopy.